Biol3100 Revision

[Auto-generated transcript. Edits may have been applied for clarity.]

So I'll just briefly go over some of the things for module two then.

Um, Carl I think will only be able to join from about half past.

Um, so. Presumably, if he does join, um, [INAUDIBLE] go over module one after that.

But yeah, feel free to pop any questions either in the chat or just unmute yourself and ask anything.

Um. Okay, well, hopefully you get your audio sorted soon.

Um. Okay.

So the second module was about infection.

Disease. Um, starting off talking about our immune system.

I'll just quickly go over that and. Yeah, stop me if you want me to go over anything in more detail.

But we got to more considered the two arms of the immune system,

the innate immune system that you're born with and is just as effective every time you see the same infection.

And then there's the acquired what you mean system, which is supposed to learn and get better each time you see an infection.

So if you've seen an infection once, then your acquired immune system should be much better at fighting it off a second time.

And these are sort of the summaries of the things involved in those immune systems.

So your innate immune system is everything from the physical barriers of like

your skin or mucous membranes to proteins going around in your bloodstream.

And your acquired immune system is largely cell mediated.

So you've got your T cells and your B cells. Um, and it provides memory and can discriminate between your own cells and something invading.

So this is a quick summary of all of the things, um, that are involved in that.

Uh, innate immune system. Um, and I won't go over all of these in any much more detail apart from, um, the complement system.

Uh, but if you do want any of these to go over in more detail, just let me know.

So the complement system is.

An enzymatic cascade that is largely mediated by proteases.

So there are over 30 proteins which are largely produced in the liver and get circulating in your bloodstream,

which are called the complement proteins. Um, and the idea of complement is that it's supposed to be activated by the presence of.

Invading pathogens, particularly bacterial cells and.

They there were three sort of pathways leading to activation of that complement.

And we'll go over those three. But they have the same basic outcome, which is to oxidise invading pathogens.

So what that means is that they get marked as.

Cells which are not supposed to be there, and they are then recognised by other.

Cells in our body which phagocytosis them and kill them.

That's what optimisation is. So all of these pathways lead to the same outcome which complement activation which is.

Optimisation. And it all comes down to that key component C3 which gets.

Cut into two parts called C3 A which is a soluble part.

And then C3 B and C3 B binds onto attaches to the surface of cells and is there as a signal saying this is an invading cell.

This is something to be phagocytosis and killed. So the first of those pathways is the alternative complement system.

So in this system, um. You get cleavage of the C3 protein?

Um, in fact, in all of the systems, you get cleavage of the C3 protein by what's called a C3 convertase.

And this is the enzyme that cleaves C3 into the C3, A and C3 B in the case of the alternative complement system.

C3 is part of that convert phase. So C3 can bind to another complement protein called B.

And B gets activated by a protein called D and get that gets cut to give you baby.

And then the other portion B again just floats off. Now in the presence of b c3 you can then cut itself.

And so C3 cleaves itself and becomes c3 b b b.

And the C3 P complex cuts itself when it's detected a bacterial surface that it can attach to.

And then the C3. BPP becomes an active converter, so it will find other C3 protein in solution and cut it.

And so it will make more and more C3 be on the surface of the cells.

And so one molecule of c3 b b b, being an active converts,

can cut many molecules of C3 and lead to studying of the surface with hundreds of potentially thousands of these molecules.

And again, the presence of all of these C3 bees on the surface is the optimisation signal.

So the second pathway is the mannose binding lectin pathway.

And this is triggered by proteins in the blood which recognise certain sugars which are only present on the surface of,

for example, bacterial pathogens. So you can see a load of those proteins here.

So the mass proteins and they will bind to these sugars.

And um then recruit another couple of complement proteins.

So they recruit C2 and C4. And both of these again get um proteins into their active forms.

So C2 gets cut into c2 a and c2 b goes off and c4 gets cut into c4 a and b and c4 a goes off and c4 b then makes a complex with c2 a,

and this attaches to the surface of the cell. And this is now the C3 convertase that's used.

So. This recognition molecule is just there to recruit a C3 converts,

and then that single C3 converter is can again chop multiple C3 in solution leading to C3,

b studying the surface of that cell, and C3 A going off as a um, cytokine signal.

The third pathway quite similar is the classical complement pathway.

Um, what that difference is, is that here you've got an antibody which has recognised some pattern on the surface of a invading pathogen.

And then this set of proteins C1, Q, C1,

R and C1 is bind to the antibodies and then recruit the same C3 converts so C to a C4 bay onto the surface of the cell.

So. In this pathway, it's recognition of certain sugars which activate it.

And in this pathway, it's recognition of antibodies which are found on to the pathogen which activates it.

But it's the same converters that's recruited the C2 AC for B.

And again that converts the C3 into C3 B and C3 I.

And one molecule of this can convert lots and lots of C3.

So they all lead to the same outcome which is lots and lots of C3 b studying the surface of the cell.

And so then that's an optimisation signal which says to the phagocytic cells to envelop that and kill it.

But it can also complement can also kill cells more directly.

And that's the recruitment of what's called the membrane attack complex.

So. The C3 b, which studies the surface of the cell, can also, um, recruit some other proteins.

So one of the ones it interacts with is called C5 B.

And then that forms a complex with C6 C7 C8.

And then multiple copies of C9. And what these things do is they're all membrane binding proteins.

And so they start to align next to each other and they form basically an open channel in the membrane.

So you can see that happening here. So you've got lots and lots of C9 forming an open channel.

And it basically has a little hole in the membrane. And just all of the internal contents can flood out.

And obviously if you have that then the cell dies.

Also allows lysozyme to enter that hole, so it gets into the Perry plasmid space and can chew up the cell wall,

and without the cell wall, the cell will explode. So different ways that it leads to killing of the same.

That all? Okay. Anyone want me to go over any part of that again?

Okay. Okay. If that's all good. Um.

So. We went over inflammation a bit.

So inflammation is a. Key part in recruiting, um, cells to the site of injury.

Um, and in the short term, inflammation is really good.

Long term inflammation is really bad.

So what happens in information is that you get these pro-inflammatory cytokines which signal to all kinds of nearby cells.

Um, and one of the things that they do is that they signal to the blood vessels surrounding their to become leaky.

And one of the effects of that is that it allows fluids to come from the blood vessels into the place where you've got an injury,

which is why after an injury, you you'll get that swelling around the site of injury.

And it also means that cells from your blood can start to come out into the side of injury.

And so what happens is that you've got lots of neutrophils going around in your bloodstream.

But now not many neutrophils reside in the tissues.

And so to get the neutrophils from the bloodstream into the tissues where they can phagocytosis cells they need those leaky blood vessels.

And what happens is that they roll across the surfaces there.

So that in another response to damage is that they start the cells lining the blood vessels start to produce select things which are sticky molecules,

so that the white blood cells will stick onto them, and they start to roll across the surface of different sticky surfaces until they can find a gap.

And then they move through that gap and into the tissues where they can do their phagocytosis.

So there are all of these other cells in your blood. By far the most common are the red blood cells.

And they're basically there just to transport, um, oxygen around your body.

You've then got, um, all of the white blood cells. And the most common of those are the neutrophils.

So the neutrophils are really phagocytic cells. Um and they are the ones which actually go into um.

Tissues and actually gobble up an invading pathogen. And you've also got um.

T cells and B cells. And so those are parts of your adaptive immune system.

So just to go over the process of phagocytosis.

So, um, phagocytic cells can recognise invading pathogens by a number of mechanisms so they can directly recognise antibodies which are bound to them.

They can recognise the C3B protein which is that optimising signal.

And they can recognise what are called um pathogen associated molecular patterns or Pams.

So these are just surface markers which are common to various different pathogenic bacteria,

which can be directly recognised by surface cell markers on some of our cells, no matter what they use,

if they can recognise an invading pathogen by any or potentially all of those means at the same time,

what happens is that they will bind to those and then start to envelop them into the cells.

So they basically start for treating their membranes to surround that invading cell.

And you can see that here. So the membranes go out and they start to surround those, um,

invading pathogens until eventually you get a membrane surrounding all of those which is now internalised into the cell.

So that's this, um, lysosome. And inside the cell, it then fuses with other vaccines in the cell,

which are filled with chemicals and enzymes, which will kill and break down that particular pathogen.

And then most of the debris of what's broken down after cells have been, um.

Destroyed is, um. Just.

It's thrown out of the cell, and so the vacuole will again fuse with the membrane on some side of the cell, and the debris just comes out.

However, the cell can also do something else, so it can take parts of that invading pathogen that it's chopped up.

And usually these are little bits of protein. So they've been cut by proteases.

And then now just fragments random fragments of protein from an invading pathogen.

And what it can do is it can take those little fragments and then present them to the immune system and say,

if you see a fragment of this protein, it's come from an invading pathogen.

So start to make an immune response against that.

And that's what these things are on the surface of the cell.

So you've got what's called the MHC class two or the major histocompatibility complex class two.

And they take little bits of chewed up protein and present them to the immune system.

And those little bits are called antigens. And so it's saying this is something that we've destroyed.

If you see this it's part of an invading pathogen.

So start to produce an immune response against it.

And the cell which produces that major immune response is the base cell.

So the cell. Um. Is called B because it matures in your bone marrow.

So be for bone marrow and it starts to produce antibodies.

So that's what these are. So antibodies have a Y shape.

So they're um effectively you've got a what's called a heavy chain.

So the longer one here and a light chain and they are connected by disulphide bonds.

And you get two of these. So you've got two heavy chains and two light chains making this sort of y shaped, um, formation.

And at the end of each of those, so where the heavy and light chain meet, you get basically a binding pocket.

You can think of it as two cups on the end of that. And each of those cups is designed specifically to recognise a particular antigen.

And so it can bind on to a particular antigen with really high affinity.

Um, but it will. It's also very specific.

So, um, that antigen might be a string of just six amino acids or so.

And if the, if it's an exact match to those six amino acids that combine to it, if there's a mismatch, it won't mind to it at all.

And what happens is that each of the best cells in your body, as they mature, they go through a process where they make different antibodies.

Um, and we don't really need to go into the fairly complicated process of how they do it,

but they do all kinds of rearrangements of their DNA so that each one, each B cell in your body is potentially.

Producing the ability to recognise a completely different antigen from every other B cell in your body,

and so you're randomly producing cells which can recognise a random selection of antigens.

And most of these never do anything in their lifetime. And they will hang around in your body and eventually die off without doing anything.

However, if one of these happens to encounter the antigen that it's, um.

Been adapted to recognise, then it gets activated and if it's then Co activated with a T helper cell,

it will then start to produce these antibodies not studded into its membrane but actual soluble versions of it.

And it can produce thousands of these which go out into your bloodstream and then will,

uh, attach onto the surface, um, or wherever they find an antigen.

Usually it's on the surface of a cell. And then they will mark that sell out as being something for the immune system to kill,

either by optimisation or recruitment of through the classical complement pathway.

Or even sometimes by just so many, um,

antibodies binding onto the surface that that cell can no longer attach to anything or come out its pathogenic function.

So that's what B cell state. But B cells in order to get activated.

Actually need a signal from a T cell. So, uh, you've got a dual level of control.

So you need the antigen in the B cell. And you also need the corresponding T cell to tell that B cell.

Yes. It's a real response that's required. And so the T cells that do that are called the T helper cells or the T cells.

And again similarly to the antibodies they've got a receptor molecule which is individual for each clone of T cells.

And again it can recognise a specific antigen.

And if it recognises the same antigen as that B cell then the two things communicate with each other and activate.

And it's this complex, the T cell receptor along with CD4 that responds to that MHC class T.

So those phagocytic cells. Presenting peptides.

Um. They are presenting them to T helper cells.

And so the T helper cell binds onto it through the T cell receptor.

The CD4 then anchors it in place. And then that's an activated T cell.

And it then goes and finds its corresponding B cell and activates that.

The other flavour of T cell is the site of toxic TSL ATC cell, which also has a T cell receptor.

But it's got a very different Co activator molecule. Create and create won't recognise a major histocompatibility complex one two.

Rather it recognises MHC class one. So that's sort of summarised here.

So um a antigen presenting cell.

So something that's phagocytosis and chewed it up will produce antigens on MHC class two those activated T cell.

The activated T cell goes off and finds a B cell. It then activates that B cell.

The B cell produces antibodies. And you also produce memory B cells.

So the memory B cells are a subclass which go off and survive for quite a long time and give you that active memory.

So the second time that you, um, might encounter that pathogen, you got a much more rapid response.

Cytotoxic T cells recognise antigens in MHC class one, and if they recognise that antigen and get activated, they will basically kill that cell.

And so usually what's presented in MHC class one is for example, viral proteins.

So if a cell has somehow detected its being invaded by a virus, it can again chop up bits of viral protein,

present them on MHC class one, and effectively commit suicide by getting it a T cell to come in and kill them.

And that is a way that your cells can stop the viral spread to surrounding cells.

And so that's the basic roles of the T helper cell and the cytotoxic T cell.

And that's just summarised here. Um, and the different effector molecules.

Um, activate them. So either MHC class two or class one.

So that's a quick summary of the immune system.

Any questions about that? Yeah.

You can go ahead, either type it in or just unmute yourself and ask.

So the question is what type of cells have MHC class one.

And the answer is basically every single cell in your body.

Um and usually what happens is the MHC class one.

As you're developing, it's presenting bits of your own.

Cells proteins. And it's it's used in training your immune system to be able to recognise your cells.

And so your immune system and again I, I'm not exactly clear how this works,

but during development your immune system recognises things which are commonly

on MHC class one as your own and therefore doesn't react to your own antigens.

And so that's MHC class one is also important in recognising self from non-self.

So then after you've developed, then anything new presenting what MHC class one has to be something foreign.

And so you then start to develop that, um, response to something novel on MHC class one.

And so again it's slightly complicated, but all cells have the ability to present things on MHC class one.

And they are basically always presenting self.

To show that they should be there. Um, it's a bit complicated, but yeah, I hope that answers your question.

The cytotoxic. T cell do you mean?

So. You know, cytotoxic T cells are, um, these cells which get activated by, um.

An antigen in MHC class one. So they've got the CD8.

So they are the T cells. Um, and once they get activated, they become what are called cytotoxic T cells.

And they are packed full of, again, enzymes and reactive species.

And they basically can then spit those out onto a cell that they've detected is.

Abhorrent and they will then kill that.

So that action of those enzymes and reactive species.

And it's one of the ways that your body is thought to basically clear any cell that looks as though it's becoming cancerous.

So if something is becoming cancerous, they might be presenting or producing proteins which are aberrant,

and those start to get presented on MHC class one. This creates cells recognise those as unusual and kill those cells.

And it's a way if you don't have that you then become very much more prone to cancer.

Um, but they also are involved in detection of viral infections or even intracellular pathogens, which can be bacterial cells.

So that's what they do. They they recognise aberrant cells and kill them.

Um, is MHC class two specific to phagocytic cells or are they present?

So many cells are MHC class one. They are especially abundant in the phagocytic cells.

And there are two types of phagocytic cells which are extremely good at presenting them.

So neutrophils are very phagocytic but they then don't communicate to the rest of the immune system.

So you've got macrophages and dendritic cells which are phagocytic and then are very good at taking antigens and presenting them on MHC class two.

It's not to say that those are the only ones that do it, but those are the major ones involved in doing that.

Any other questions? Yet this whole, um, lecture should be recorded on canvas.

So going on to, um, disease.

Um, if there are more questions, feel free to put them in the chat, um, or unmute yourself and just ask.

Um, so when talking about the disease, one of the things that is always of interest is how they actually get into your body.

So the skin to most pathogens, uh, microbial pathogens is impermeable.

And so they need to get in through some other way.

And usually that's either through a, um, lesion in your skin so that they can get straight into your bloodstream or into tissue or through invading,

um, colonising and invading, um, mucosal membranes.

So you've got mucosal membranes pretty much any time that your body needs to encounter the outside world.

So in your gastrointestinal tract, where you've got to interact with food in your lungs,

in your genital, urinary tract, um, all covered with these mucous membranes.

And they are portals that pathogens could get into the body.

Um, you've also got things like biting insects which can bypass the skin and so on, surgical site wounds and so on.

So that's sort of summarised here. So injuries needle sticks anyway where you've got open wounds um contaminated water.

So ingestion is a very common, uh, mechanism getting into your body.

There are direct contact diseases. So sexually transmitted diseases and so on.

And so it's important to recognise the different ways in which things get into the body.

And that's also important in the way in which they then can cause harm as well.

So for example, uh, Vibrio cholerae, um, only colonises your gastrointestinal tract.

So if you only get cholera from ingesting usually contaminated water,

if you've got an open cut and you put that into contaminated water, you're very unlikely to develop cholera.

Um, because the vibrio won't be able to usually migrate from an open cut into your GI tract.

And they they're not adept at colonising fresh wounds.

So. What marks out a pathogen from, um, any other sort of benign bacteria is the ability to colonise a host and then cause damage.

And so.

Any of the factors that a pathogen produces which help in either colonisation, getting in persistence or causing damage are called virulence factors.

And so if you think back to the assignment two, where you looked at Clostridium difficile, it's got all kinds of different virulence factors.

So its ability to form spores, which are highly resistant, is a key virulence factor.

And that allows the Clostridium to bypass your stomach acid, which would normally kill most bacteria.

But the spores are so resistant they get through your stomach and then they can germinate in the more alkaline environment of your intestines.

And so that's counted as a virulence factor.

Then the ability to say, um, attach onto mucosal membranes and then form a biofilm, that's another virulence factor.

Um, and then being able to directly cause damage either through projects or metabolites or toxins is another virulence factor.

And one of the key types of toxins that we went into are the ab Xa toxins.

And there are lots of important examples of these. And it's important to understand how they actually work and cause damage.

And so they they work by having two proteins which join together in a complex.

And they are called a and b. And the b part is like the delivery system.

So the b part is usually um binds onto some receptor on the cell surface and allows um by different methods,

but usually by m um and dose high doses to get the AB complex into the cell.

So you get andesite ptosis forming a vesicle inside the cell with the AB.

This then gets um acidified um, and then uh, the A and the B part can part ways and the a part can come out of the vehicle in some way.

And then it goes into the cytoplasm and then does its damage.

So the uh, example shown here I think is from um.

I've forgotten the name of that disease. Uh.

Diptheria. That's it. So it's the diphtheria toxin which ADP rib oscillates.

Elongation factor two, which is important for translation.

And so you no longer get any translation in the cell types.

But there are all kinds of different ways in which these ab toxins work.

Um, so it's good to know examples of this and understand how they work and how an exo toxin.

Differs from an endotoxin. So endotoxin is part of the actual cell itself, of part of the pathogen.

Usually what we think of as an endotoxin is the lipopolysaccharide on the outside of gram negative cells.

And so when the cell dies, it can just release lots and lots of that LPs.

And that helps is actually incredibly toxic and causes a huge immune response.

And so we went over in the lectures. Examples of viral diseases, bacterial diseases, fungal diseases and protocell diseases.

And, um, it's probably worse for the exam knowing examples of each.

Um, and. What we want.

Usually in exam questions like in the um.

Tutorial questions is some level of detail.

So what the causative agent is, what disease it causes, how it gets into the body.

So it's portal of entry. What are its virulence factors.

So um how does it colonise.

What toxins does it produce. And then what are the symptoms of that disease.

So um, what's the effect of those toxins and all the other things.

And so knowing some details about, um, examples of these, um, would put you in good standing for the exam.

We then also went over antibiotics and antibiotic targets.

Um, and I'll go through this fairly quickly and they can be broken down, um, basically into uh, what's shown here.

So cell walls, cell membranes, nucleic acid production, protein synthesis, um or biosynthetic pathways in the cell.

Um, and so understanding and maybe having an example of a, uh.

Antibiotic which might do these. And understanding how they work.

Um, at least to some extent would be good. Um.

This is again. I've included this just for your revision.

I won't go over this in any more great detail.

It just sort of summarise is all of the different types of antibiotic which all target the ribosome and so inhibit translation.

So all of these different classes inhibit slightly different steps of um and of the translation process.

And then we went over drug resistance. Um, so there are multiple ways in which you can become resistant to these drugs.

So there's basically, um, not allowing the drug inside the cell in the first place.

So either altering the composition of your membrane or, uh, down,

regulating the portions that allow things in, or you can efflux the antibiotic straight out again.

You can mutate your target so it's no longer recognised by the antibiotic.

You can modify the target so phosphorylated or methylated.

So that again the antibiotic won't be able to bind. You can uh overproduce the target mimic um and so on all of these different mechanisms.

Um, and as long as you understand generally that there are lots of different mechanisms and you've got some examples of them,

then that would be great. And again, this is a really detailed, um, slide.

Um, and you can just look through that while you're revising and see if you can understand, um, all of the things that's going on there.

And it's quite useful that it sort of emphasises what's a gram positive and what's a gram negative.

And some of the different, um, the ways in which antibiotics might work in one class and not the other.

And then the final thing we went over in um, module two was epidemiology.

And so in epidemiology what you're doing is looking at so the natural history of an infection.

Or series of infections. And so what epidemiologists try to do is identify what's causing a particular illness.

So can we identify the agent? Is it a bacterium?

Is it a virus? Is it a fungus? Um, how is it getting into people?

Where's it coming from? Is it people coming from a contaminated well?

So there's a common source or is it person to person transmission?

Um, how's it getting if it's person to person? How's it getting out of people?

Um, mode of transmission and so on. How's it getting in?

What? Makes some people susceptible.

Other people not. Is it affecting everyone? Um, are there certain people who are affected more than others?

And generally speaking, it's people with poor immune systems who get more affected.

And those are typically the very young and very old, but also people with immuno deficiencies.

And there were some, um, illnesses which can infect and affect, uh, perfectly healthy, um, adults as well.

And so, uh, understanding that sort of multiple links in the chain of infectious cycle is important

so that you can break that infection cycle by cutting any of those links in that chain.

For example, if you've identified a reservoir, can you remove that reservoir?

So if that reservoir is a contaminated water source,

can you basically remove people from going to that water source if that reservoir is mosquitoes carrying it?

Is there anything you can do to either eradicate the mosquitoes or at least control their numbers and so on?

And so again, you're trying to see whether you can at least understand the infectious cycle,

um, follow it and then give advice to governments on how best to control it.

Um, and some of the, uh, terminology that you need to be, um, familiar with.

Uh, what is an outbreak? What's a sporadic disease?

What's an endemic disease? Hyper endemic epidemic and pandemic.

And those are the definitions. They're. So I think that's basically everything from module to covered.

Does anybody have any questions from that. Not seeing anything yet.

So. Um, I might hand over to Carl if you're ready.

Um, if you want to. To, um. Anything from.

Module one. And then I guess if we have time, we'll go over module three after that.

Yeah, I've got slides from module one and module three. Um, I'll try and be quick with module one, um, so that we can get to module three.

Um, I definitely have too many slides. Um, uh, hopefully, uh, I can skim through some of it.

Is that. No, I'm not sharing it, am I? Okay.

No issue with them. Uh.

Yeah, that's still in present mode for us. Uh, okay.

And, uh, what to exclude? Yep.

That's cool. Um, okay. I'm having some issues with images being lost.

Uh. That's a bit weird.

I think this is the one. The other shape. Uh.

It all. Appearing in.

That. Sorry.

Um. If you want to sort those out, I could do my bits of module three while you sort that.

Or, uh, you. Um, I can, unless you think it's going to be a very quick fix.

Uh, I can try. And say, okay, here we go.

We're gonna try again. You can't better.

Yes. That looks good. Okay. Okay. Okay.

Um, so in module one, uh, I guess the first goal of module one was to illustrate, uh, the level of diversity,

uh, within the microbial world, uh, particularly within prokaryotes, which were, uh, the primary focus.

And, uh, as we can see from the tree of life, when we get to it, it's certainly probably the dominant lifeform on Earth.

Uh. Just trying. They've got. Part of my screen is obscured by.

Okay, here we go. Let's collapse then. And then I can't see myself, which is good.

Um, so we started by talking, um,

a little bit about microbial taxonomy and the tree of life and how we classify micro organisms, uh, into that tree of life.

Uh, so at the minute, we recognise three domains of life.

And so these are, uh, the eukarya or eukaryotes, um, the archaea and bacteria.

Um, and so historically, uh, care in bacteria, uh, because of their morphological similarities,

um, had been grouped into two one groups, uh, into the prokaryotes.

Um and the eukaryotes were separate. Uh, but as we got better at classifying organisms, we moved, um, to three domains,

because we could recognise that bacteria and archaea were indeed extremely different.

Um, so that was based on, um, molecular analysis.

Uh. So, um, this table basically summarises the different approaches that scientists have taken, uh, in the past.

And now to classify organisms, um, into different taxonomic groups.

Um, so we've got phonetic classification systems, uh, which basically look at phenotype of organisms.

So for example, with baseball form spores or the morphology of the organism, um, genotypic,

which looks at genes and genomes, um, and um phylogenetic where we also look at these genes in genomes.

Um, but we use um models to try and understand how these organisms have evolved.

So based on rates of change of nucleotides, um, and so on, to build up, um, better pictures of how closely related, um, different organisms are.

Uh, one of the major, um, advances in understanding microbial taxonomy, of course,

came from the work of Carl was, uh, so in the late 1960s and 1970s, um,

and basically what he and his colleagues did was to recognise that, um, slowly changing, um,

sequences that were conserved across different organisms could be used, uh, to classify them.

So kind of like a molecular clock. Um, so we expect some random changes in DNA over time, um, just through mutations.

So they're important for generating diversity in organisms.

Um, if we've got highly important sequences like the Robbins animal RNA sequences or small subunit ribosomal RNA sequences, uh,

we can look at differences between those sequences in different organisms to work out,

uh, approximately, um, how, um, distantly or closely related they are.

Um, so based on his work, um, in those principles, we've been able to build, uh, quite detailed trees of life.

Um, and so this is still one of the best examples of those.

Uh, this tree was generated by Gillian Banfield, um, who's, uh,

Australian originally now living in California, um, at UC Berkeley, um, where she works.

Um, and what her group did was to get sequences.

Um, so for most of these sequences, they came from the environment.

So she was using metagenomic approaches to isolate those sequences, um,

and then use them to classify organisms into this, um, fairly comprehensive tree of life.

Um, and so what we can see in this tree of life, um, is the domain structure, uh, that we were talking about earlier on, but in a lot more detail.

So at the top here, all of these branches represent, uh, bacteria.

So different groups of bacteria. Um, down here, we've got the archaea, um, and then very closely related to the archaea, uh, the eukaryotes.

Um, so we know that, uh, cator and eukaryotes have, uh, a common ancestor that,

um, is occurred in, um, less distant past than the universal common ancestor.

Um, so bacteria diverged from these groups, um, earlier.

Um, in this figure, each of these dots shows a group that we haven't got information for.

Like we haven't been able to isolate those organisms. Um, and so you can see that a lot of the tree of life, um, is kind of unknown.

So there's a huge amount of diversity out there that we haven't been able to culture, um, or isolate.

Um. I always find it fun to look on these trays.

Um, actually. So these little red ovals that popped up, uh, the groups that we looked at through module one.

Um, so we took a whirlwind tour of the the tree of life of the tree of Cellular Life.

Um, yeah. It's interesting to look at where humans and or animals and plants sit on this tree of life.

Uh, because often when we talk about life, those are the things that pop into our heads.

Um, so we've got all animals sitting down here on a branch, along with fungi.

Um, in the context, um, and all plants, uh, fit into another small branch here.

So the access data, uh, along with algae. Um, and so when you take out plants and animals, um, the rest of the tree of life is microbial,

such as the massive diversity, uh, that we have in the microbial world.

Um, and it's responsible for a huge, um, range of functions on Earth.

Um, of course, this tree doesn't include non cellular life forms.

So things like viruses, um, and virus related, um particles and browns.

Um, so there's even more diversity that we would consider microbial uh, which is not shown on this tree of life.

Um, so those trees are basically, uh, based on this hierarchical taxonomic system.

Um, so at the very top, we've got our domain. So bacteria, archaea or eukarya, um, sitting under those, we have different phyla.

And so a huge number of different phyla that have been identified.

Uh, particularly with the use of, uh, metagenomics and so on, where we don't need to culture organisms.

Um, then we have class or family, um, and genus and species.

So, uh, through module one, you would have heard a lot about different, um, general, um, Genesis, um, uh, and species, uh, and also different phyla.

Um, so I think we basically tracked through different filer and gave some examples of, uh, the types of species that exist within that,

um, phylum to give you a diversity, uh, give you an idea about how much diversity there is even within, uh, each phylum.

Uh, we don't need to get into that too much.

Um, so, yeah, another point of module one was to try and illustrate how abundant microbes are.

Um, so we know that there are, uh, at least ten to the 30, which I think is ten nonillion, um, prokaryotic cells estimated to be on Earth.

Uh, which is more than there are stars in the galaxy.

Uh, and there's an order of magnitude, at least more viral particles than that.

So, uh, for every, uh, prokaryotic cell, there's probably ten virus particles, at least.

So huge. Um, huge abundance. Uh, and they're also very metabolically diverse.

Um, so they use different energy sources. Um, they have different carbon sources and electron sources.

Uh, we did cover a lot of this in um Biol 2019.

Um, but ultimately, because of their abundance and their metabolic diversity, um, prokaryotes can have a huge impact on the world.

So, uh, particularly in biogeochemical cycles.

Um, so moving carbon, um, and nitrogen and so on from organic to inorganic forms.

So they're a huge force. Um, and potentially one that we can harness into the future, uh, to fix some of the issues that we've created.

Um, tipping the balance of those biogeochemical cycles in the wrong direction.

Uh, so after our initial lecture, um, we started to go through different prokaryotic groups.

Um, and this is, this was done.

Uh, basically according to, uh, the textbook, um, which for historic reasons, um, followed, uh, a scheme from Verghese Manual,

uh, which was, uh, an old, um, manual which basically outlined the types of prokaryotes that exist.

Um, uh, um, so some groups aren't necessarily grouped together for, um, any particular reason other than convenience.

Um, and that's because in Verghese Manual they were groups together, um, to basically put together chapters.

Um, so we started talking about archaea.

Um, and so these are kind of interesting because we know that they share, um, a common ancestor with, um, the eukaryotes, uh,

their last common ancestor, um, is more recent and the last universal common ancestor of all organisms when we look at these traits.

Um, and so because they have some, um, relationships with eukaryotes, uh, we do see some similarities.

So for example, in cellular functions like replication, transcription, translation, um, they also have some features in common with bacteria.

Um, so they morphologically similar to bacteria, um, and some of their metabolic processes are also related to the ones that occur in bacteria.

Uh, probably because they are small single celled organisms, um, and they absorb most of their nutrients, uh, from their environment,

or they have to absorb nutrients from their environment and the types of things that they can access,

uh, similar to the ones that bacteria can access. Um, but there's also unique elements to the archaea.

Um, so, for example, they were put into their own domain because they've got their own take, um, rather than normal RNA gene structure.

Um, and some archaea can, um, mediate processes that we don't see in other domains.

So for example, kind of Genesis is something unique to the archaea.

Um, they are of course highly diverse. Um, and you could see an indication of that diversity from the tree of life.

Um, by comparing them to what the diversity that we see, uh, for example, in animals and plants.

Um, the tree of life illustrates that, uh, the different groups in the archaea, uh, a lot more diverse than those, at least genetically.

Uh, we looked at some major, uh, Keele groups.

Um, and I won't go over these in a huge amount of detail.

Um, something to remember is that a lot of them are extremophiles.

Um, so they live in extreme, extremely thermosphere like environments, um, and can make a living in that.

Um, typically as, um, by oxidising, um, nutrients that are available there so that we often see sulphur oxidation, for example, in those DNA files.

Um, over time, uh, our understanding of the archaeal tree of life and, uh, archaeal taxonomy, um, has developed quite significantly.

So we started out with three phyla when Boogie's manual was first published.

Um, and you can see those listed down here. They were the Europeana chronology.

Ida and Thelma Quijada. Uh, but now we recognise that there are multiple super phyla.

So containing within them, um, each multiple phyla.

Um, and there are also some new suit phyla.

Um, so one of particular note, uh, the old guard um, which were only identified, um, in the last ten years.

So only in um, 2015. Um, and these are interesting, uh, because, uh, they contain I'll skip over this.

They came from these thermal vents, um, under the ocean.

So up in the Arctic, um, they interesting because, uh, we see features in the old archaea, um, that we would typically associate with eukaryotes.

Um, so they contain subcellular structures and genes for subcellular structures.

Um, that. Sorry. I'm jumping around a bit because of my mouth, um, that we would typically find in eukaryotes.

So we've got trafficking machinery for example, um cytoskeletal proteins um, which we would typically otherwise only find in eukaryotes.

So we don't find them in these other archaeal groups. Um, a lot of them.

Um, but we do find them in archaea.

Um, and so this raises the possibility that these were, um, missing links or the closest living relatives, uh, to eukaryotes.

Um, and when we look at the morphology of some of these cells, so some of them have been isolated, uh, they do kind of look eukaryotic.

So they have, uh, projections in them that reflect the presence of a cytoskeletal complex, other skeletal, um, proteins.

Uh, and I won't go into that. Uh, but this is basically illustrating potentially how eukaryotes evolved from these primitive archaea.

Um, so perhaps these, um, complex skeletal proteins and these projections allowed them to form intimate interactions with surrounding organisms,

like, for example, alpha proteobacteria.

Um, and then over time they developed um and obligate um symbiotic relationship where they relied on one another.

Um, and then ultimately, um, those bacteria may have been internalised into those primitive archaea,

um, forming the um, mitochondria that we see in all archaeal cells today.

Um, and based on some of this research, um, it's now been, um, suggested that perhaps eukaryotes,

uh, um, basically a part of the archaea, um, and so they sit within the archaeal domain.

Um, and based on that, we, uh, potentially moving back to, uh, two domain view of the tree of life.

So we originally recognised prokaryotes and eukaryotes, um, as two separate sort of broad groups.

Um, then with molecular taxonomic taxonomic methods, we were able to identify the archaea were quite different from the bacteria.

And so we currently recognise a three domain tree of life.

Uh, but with this new information, uh, perhaps archaea, uh, forming part of uh, um, or perhaps eukaryotes are forming part of the alcohol domain.

Um, so potentially moving back to a two domain view of the tree of life.

Uh, we then went on to look a little. A little bit of photosynthetic bacteria.

And so we see these sitting, uh, in different groups across the bacterial domain.

Now. So we've got purple non-social bacteria within the, um, Proteobacteria.

Uh, green sulphur bacteria growing on sulphur bacteria.

Um, and so in our bacteria, uh, which is what we typically think of when we think of photosynthetic bacteria.

Um, and these are definitely, um, the most abundant photosynthetic bacteria, um,

and the precursors we think of or plants, almost certainly the precursors of chloroplasts.

Uh, so these uh, obligate or typically obligate, but, uh, the autotrophs, um, some of them can metabolise pre-formed organic carbon.

Um, indeed, they generate organic carbon, um, during photosynthesis.

Um, and they can metabolise that. Um, they do that by oxygenic photosynthesis.

Um, so oxygen is generated, um, as electrons are donated from water, um, using energy from sunlight, um, to split and release those electrons.

Um, some species can fix atmospheric nitrogen, um, and convert it into organic forms.

Um, and so they're important both in carbon cycles, oxygen cycles and nitrogen cycles.

Um, through these different processes that they can mediate in their metabolism.

Um, we then went on to look at some other groups.

Um, I'll go through these kind of quickly. Um, for the exam, I guess.

Um, something you might be asked, uh, is to compare some of these different groups to one another.

Uh, with respect to the impacts on, for example, the environment or on health.

Um, it's a it's good to know, uh, within each of the groups,

maybe 1 or 2 examples of different species that are able to mediate different processes and might impact different,

um, environments or have an impact on humans in different ways.

And so we started looking at the spa kits and tannery kits.

Um, and as you can see, there's, uh, very different groups of organisms, um, grouped together for convenience.

Um, in Bergen's manual. So one of those examples, um, the, the tannery kits.

Um, basically this translates to um, tend to cutis, uh, tend to skin.

Um, and this is basically because they don't have a cell wall.

Um, so we've got organisms like mycoplasma, um, which, uh, obligate or generally obligate parasites.

Um, so they're able to exist without a cell wall because they exist inside another cell, which has a very osmotic, be stable environment.

So the cell wall, um, particularly the peptidoglycan layers within the cell wall, uh, protect the cell from osmotic stress.

Uh, they like to concentrate nutrients inside the cell. Um, and that can cause them to rupture.

Um, if they're not in an osmotic stable or a stable environment, um, because they don't have a cell wall,

um, when they're cultured, they can have this clear morphic apology.

So, um, so wall quite often will dictate the shape of the cell.

Um, and if they don't have it, then they can grow in all sorts of different shapes.

Uh, one interesting example of these, uh, is a purely synthetic mycoplasma.

Um, so because they're obligate intracellular parasites, they've got small genomes.

Um, and that made them appealing to researchers who were trying to completely synthesise bacterial genomes, um, using chemical approaches.

Um, so the first few examples of completely synthetic microorganisms or organisms in general, um, were of mycoplasma.

Um, and this was done by J. Craig Venter and his colleagues.

Um, I'll come back to this very briefly. Uh, when we talk about module three and other organisms that have had their genomes completely synthesised.

Uh, we also looked briefly at the spire case. Um, so these have got the characteristic cell morphology, uh, which is kind of like a corkscrew shape.

Um, they interesting because they, um,

are able to move using a flagellum which sits within their Perry plasmonic space and kind of wraps around the cell.

Um, so as they turn that flagellum, um, rather than it acting like a little propeller,

it basically causes the whole cell to, to corkscrew along waste.

Um, and this is potentially important to their life cycle, uh,

because it might allow them to bury themselves and burrow down into, um, different materials.

Uh, and a unique mode of motility, uh, within the spider cates.

Um, they, uh, lots of different organisms.

Um, so some of them are free living, um, some form symbiosis.

Um, so they can live in the guts of different animals, uh, insects and larger animals.

Um, and they can help them to, um, metabolise food, foods.

Um, so in termites, for example, helping them to, um.

To digest wood and potentially fix, um, carbon.

Uh, but a lot of these are also pathogens. Um, so there's some classic examples of spark pathogens that.

Uh, we then looked at the Proteobacteria. Um, and this is a massive grouping, uh, which has huge impact on humans in lots of different ways.

Um, so you can see here in the bacterial domain, the Proteobacteria.

Um, maybe a fifth or a quarter of the diversity of the, um, the bacterial domain sits within the Proteobacteria.

Um, historically they were grouped into five, um, different um, classes.

Uh, but when we had, uh, re-evaluation of, um, the, uh, names that we give to, um,

bacteria and prokaryotes in general, some of them were reclassified into other groups.

Um, and so some of this renaming of uh, bacterial father is based on um needs from metagenomics identifying lots of new different types of organisms.

Um, but we still do recognise these major groups. So elevated gamma, Delta and ellipse on Proteobacteria.

Um, here's a nice table that summarises, um, some of the important or Proteobacteria classes.

So this time, um, in the Alpha Proteobacteria, um, some of these are highlighted because they're cool.

So here we've got the magnetar tactic bacteria.

Um, and so basically they have, um, little magnets inside them, little magnetite crystals, um, which is thought to help them figure out, uh,

which way is up and which way is down, so that they can position themselves appropriately in the water column,

um, to get an appropriate amount of oxygen.

So you can see that in micro air feel like that they've got very specific oxygen needs, uh, that they need to satisfy.

Um, some of them are not fine. Um, so they play an important role in the nitrogen cycle.

Um, so mediating important step of, uh, um, uh, converting, not trade.

I'm not trying to not. Right. Um, and then others form relationships, um, with plants.

So they're symbiotic. Um, important ones.

The, uh, the right erbium. Um, so these, uh, nitrogen fixing, uh, bacteria, which form, um, mutualistic relationships with plants.

Uh, so there's two way signalling between these bacteria and the roots of legume plants.

Um, they, uh, attracted to the legume plants and, um, become internalised, uh, and form these root nodules within, uh, the roots of legumes.

Um, and within those root nodules, they will fix atmospheric nitrogen, um,

protected from oxygen by processes in the root, uh, which can interfere with nitrogen fixation.

Um, and in return, the plants will feed them with, um, organic carbon, um, so that they can continue to grow.

Uh, we've also got nasty bacteria that form relationships with plants in the alpha proteobacteria.

So for example, agro bacterium um, which is attracted to, um, wound in root fruit plants.

Um, and again, there's um, various signalling that goes on, um,

and it transfers a piece of DNA called the T DNA, uh, which gets integrated into the plant genome,

um, and causes the plant to release additional nutrients for the agro bacterium to grow and to form these tumours, um, on the plants.

Um, of course, this is the same. This is important because it's used in biotechnology.

So it's an important way of introducing DNA into plants.

Uh, a different type of proteobacteria. So he ellipse on Proteobacteria.

Um, we've got the Helicobacter. Um. So these can be isolated, um, as commensal in human stomachs.

Uh, but they can also cause, um, disease. So we'll see, like, um, peptic ulcers.

Um, the commensal ones have been used in an interesting way to try and track the movement of humans, uh, across the South Pacific.

Um, so, based on, uh, um, phylogenetic relationships of these organisms, the researchers were able to link,

uh, where and when humans sort of migrated through, um, the South Pacific.

Um, in parallel with this study, there's also been linguistic studies done, um, which came to the same conclusion, which is interesting.

Um. Helicobacter, of course, is the cause of peptic ulcers.

Um, and, uh, being Australians, we have to recognise the important work of Barry Marshall,

um, and his colleague Robert on Robin Warren, um, in identifying this association.

Um, because Barry, um, did the time honoured tradition of self-experimentation in science, um and fulfilled Coke's postulates, um, by consuming,

uh, Helicobacter pylori, um, and demonstrated that he then had peptic ulcers, which could then be cured by, uh, course of antibiotics.

A very important research. Um, how are we going for time?

I think I might need to speed up. Uh, we then looked at, uh, gram positive bacteria.

Um, so focusing on the two major groups, the actin bacteria and the Fermi cates.

Uh, within the Fermi case, uh, we've got three different classes.

So cluster, uh, uh, bacilli and negative rates.

Um, so I won't go into too much detail with these.

The negativity. It's, uh, interesting because, um, they've got a cell wall which looks more like a gram negative cell wall with an outer membrane,

uh, which we don't typically see in, um, Fermi kids apart from those.

Um, so in contrast to the tannery kits, these ones have a strong skin.

So, um, it's cutis. Uh, most of them are, uh, rod shaped or spherical.

Um, and some cause disease. Uh, a lot of these produce very nasty toxins, um, causing very nasty, um, human diseases.

Anthrax. Tetanus, botulism. Um, also food poisoning.

Um, but we have also applied some of the toxins, um, in, uh, biotech, agri biotech.

Um, as insecticides. Um, and others are important for food production.

Uh, some of these are able to form into spores.

Um, so these are basically, um, cells with, uh, or sort of dehydrated cells with a modified,

um, cell envelope, uh, which is very environmentally resilient, tough.

Um, and, and this ball production can be induced, um, in times of environmental stress.

Um, so basically it's a way of the cell making sure that its, uh, progeny are able to survive in,

in a tough environment for a period of time until, um, conditions become more favourable.

Um, in which case they will return to a vegetative state.

Uh, we looked at Clostridium. Um, so some of these are very nasty.

Um, we've got botulism, um, which causes food poisoning.

Um, and produces botulinum toxin. So Botox, um, which has been applied in medicine, um, as well as in um.

Uh, cosmetic, uh, treatments. Um.

Yeah. Clostridium, uh dificil causes gut infections.

So enteric infections. Um, Clostridium nai also produces a nasty neurotoxin.

Um, has the opposite effect to botulinum toxin.

So this this induces flaccid paralysis.

Whereas Clostridium techni um induces um.

Uh, muscle contractions. Um Clostridium origins.

Um, another nasty Clostridium which causes a number of different diseases depending on the toxins that it makes.

Uh, within the bacilli, there are two main orders are the Bacillus and Electabuzz Scilly Isles.

Um, some of the bacillus. Um, so, like the genus Bacillus, um, and the genus stuff like Orcus can include, uh, pathogens.

Um. So that's Elizabeth Rice's example of one of those nasty pathogens.

Um, and Staphylococcus aureus is, uh, an opportunistic human pathogen.

Uh, but they also include quite a number of benign species that don't, uh, impact humans in a negative way.

Um, the Lactobacillus, um, includes the genus Lactobacillus, which, of course, we use, uh,

potentially as a probiotic, but also in food production because it can to defy the environment.

Um, we talked also about a kind of bacteria.

So these are also gram positive bacteria, uh, but high GC in comparison to the Fermi case.

Um, these are interesting because a lot of them grow, um, as a filamentous hyphae.

Um, and they can also produce spores, but different type of spore, um, at the tip of the aerial hyphae.

Um, because of this, uh, in the past or like, they can be confused with fungi,

which also have, uh, a similar morphology, um, and may be similar, um, in the environment.

Um. These are important because they produce a huge array of different secondary metabolites or specialised metabolites,

which we have applied in different areas, particularly in medicine.

Um, a lot of antibiotics, but also other um bioactive drugs come from these organisms.

Uh, so this just illustrates how they're able to grow.

Um, uh, five feet. So they have a substrate, mycelium, which burrows down into the matrix.

Um, and then the aerial hyphae. Uh, and I mentioned that they produce a huge array of secondary or specialised metabolites.

Um, some of these you might be familiar with, uh, Gossman.

Um, you can smell that's basically the smell of the rain.

Uh, when it starts to rain, that's the volatile, uh, specialised metabolite released from, um, species of streptomycin, uh, in the soil.

Uh, streptomycin is, uh, in the acting and bacteria.

Um, and we've also isolated a range of other bioactive compounds from these.

Um, also in this group, uh, the mycobacterium. Um, and they are a special case because they have, uh, unique cell wall structure.

Um, so outside the pet to the glycan layer, they have, um, a waxy cell envelope, um, composed of my colleague acids.

Um, some of these, uh, um, human pathogens.

So, for example, Mycobacterium like prey. Um, and this waxy cell envelope is a problem because it's quite impermeable to small molecules.

So, um, very difficult to get things to pass across that waxy cell layer.

Um, and indeed, it's very difficult to even detect them.

Um, microscopy directly with a microscope.

Um, because it's difficult to stain them. So we need to use things like acid bath staining.

So, you know, gram stain. Um, these would basically not appear because of that waxy cell envelope.

Uh, we then moved on to a little bit of microbial ecology.

Um, and we looked at different types of symbiosis between, uh, prokaryotes and host organisms.

Um, so we talked about consortia or complex communities of microbes.

Um, Ecto symbiotes, where symbiotes, um, sit on the surface of, um, the symbiotic partner, and endosymbiosis, where the symbiote is internal.

Uh, we looked at these different types of symbiosis, uh, mutualism, um,

in cooperation, where we see two way benefit between each of the interacting partners.

Um, with mutualism being, um, more obligate than cooperation.

Uh, uh, commensal ism, where one organism benefits from the other, but the other is not harmed.

Um, predation, uh, where one organism, um, consumes or protects another, um, as its food.

So one organism is definitely harmed in that interaction.

Um, parasitism, uh, where, uh, a parasite is able to feed off a host, so to the host's detriment.

But the advantage of the parasite. Um, I'm a mentalism.

Uh, where one organism is not benefited or harmed.

Um, but another one is. So this could potentially be with the release of a waste product that is toxic to a surrounding organism.

Um, and then competition. Uh, feeling competition.

We've got two different organisms, uh, competing for the same resources.

Um, and one organism might completely outcompete another, or they might both be able to utilise that resource, but, um, at a lower level.

So they both slightly, um, it's detrimental to both, um, to some extent, uh, in nature, most microorganisms exist in biofilms.

Um, so these are basically communities of interacting organisms.

Um, typically these would contain multiple different species, um, and they have important roles in the environment.

Um, so they allow organisms, for example, to exist, um, stably on certain surfaces, um, across time.

Um, and we'll see an example of that when we look at um, water microbiology and benthic communities.

Uh, we also spoke about in lecture five, um, the impact that microbes can have on the environment.

So, for example, through biogeochemical cycling, um, and we've seen some examples of these,

for example, with uh, photosynthetic uh bacteria that can impact carbon, nitrogen and oxygen cycles.

Um, and basically they cycle elements um, through biotic and abiotic forms, uh, through their metabolism.

Uh, and this, of course, is a classic example of the biogeochemical cycle.

So the carbon cycle, um, we can have carbon, um, moving between different forms.

So we've got carbon fixation where, you know, organic carbon dioxide is fixed, um,

by a typically finite autotrophs, but other types of autotrophs which get their energy from, you know,

organic molecules, um, we see methane agenesis where we've got carbon dioxide, uh,

being converted into methane, um, and then a trophy for methane, um, being oxidised back to carbon dioxide.

Um, and organic carbon can be oxidised, um, back to carbon dioxide by our heterotrophs.

So as it's consumed, uh, and broken down. And so that's what we see through respiration, uh, with the release of carbon dioxide.

Um, then we looked at, how are we going for time?

Um. Uh, water as an environment for microbes.

Uh, and then finally soil as an environment for microbes.

So we started, um, looking at the ocean. Um, we looked at different zones within the ocean.

And so each of these, um, has, for example, different, uh, access to different,

um, amounts of light, um, and potentially two different levels of nutrients.

Um, importantly, also different amounts of oxygen and carbon.

Um, so at the surface of the ocean, at the open ocean, um, is where we see most,

um, primary production being taking place with our fur and synthetic microbes.

Um, and so because of that, we have, uh, higher concentrations of oxygen being released, um,

or we have, um, oxygen also coming from the atmosphere and, um, uh, higher levels of the ocean.

Um, we see less, uh, dissolved carbon dioxide.

Um, so some of that is being fixed during primary production.

Um, and it's solubility, um, can change with pressure.

Um. These figures basically summarise the abundance of microbes in the ocean open ocean.

So bye bye Ms. We've got a lot of prokaryotes, uh, bacteria and archaea.

Uh, but by volume you can see that there are a lot more viruses, um, in the open ocean than there are, um, bacteria, archaea or protists.

Um, and so this is important in nutrient cycling.

Um, because, uh, so most of the carbon in the ocean is being or the primary production is being done by,

um, photosynthetic bacteria in the surface waters.

Um, those, uh, photosynthetic bacteria are being predated on by those viruses.

So there's hugely abundant viruses. Um, that leads to the release of dissolved organic matter in the surface waters.

Um, and basically makes it available to other microorganisms in those surface waters as well.

Uh, because of the actions of those viruses.

Um, not all that much of that, um, organic matter that's fixed by the microbes in the surface waters is able to be,

um, consumed by larger organisms and to move up these terrific levels.

Um, if it does so, then it can get lost down into, uh, the bottom of the ocean so it can sink down into the bottom of the ocean.

So viruses play an important role in the microbial loop.

So keeping that carbon up in surface waters.

Well that dissolved organic carbon up in surface waters. Um, and also these larger organisms.

Um, their waste can also be fed along, um, by microbes in the surface waters of the ocean.

Um, and so we refer to that as the microbial loops. Uh, we then went on to look at freshwater on Earth.

Um, and of course, a lot of the freshwater on Earth is frozen.

Um, and so it's at the poles. Um, and the poles are indeed home for some microorganisms.

Um, so we refer to the microbes that live in these very cold environments as sacrifices.

Um, they also need to be able to tolerate, uh, very dry environments because all that water is frozen.

Um, and it's not in liquid form that you can use.

Um, they've also got very high UV radiation, um, which is of course seasonal.

Um, because of the, the tilt of the Earth. Uh, and there's low nutrient availability.

Um, it's extremely cold. Uh, and there are raised or cycles.

Um, so we do see microbes existing in different places in Glacier.

So some of those, uh, will exist, um, in these little, um, uh, cracking, uh, holes on the surface of glaciers and frozen water.

Um, we can get photosynthetic, um, bacteria in there like cyanobacteria.

Um, they're absorbing light. Um, and they're quite dark against the surface of that glacier.

Um, and so that absorbs radiant heat, um, and leads to a larger hole.

Um, and with those organisms in there fixing carbon,

it can also then attract other organisms that can come along and feed on that, um, organic carbon.

Now there are some microbes that we might find, um, throughout glaciers, but the abundance is extremely low.

Um, but we do find in subglacial lakes that the, uh, a lot of, um, autotrophs, um, which, uh,

kena autotrophs using, um, organic nutrients as the electron source, um, to, to fix, um, carbon.

So we see cycling and these types of nutrients down in those environments.

Um, so, uh, there's different types of, um, liquid, uh, water systems.

So we've got our systems, uh, which are free running waters.

Um, so rivers, um, streams, canals.

Um, so we've got that horizontal movement of water.

So we don't see the same stratification that we do in large lakes or out in the open ocean.

Um, because of that constant water flow.

Uh, we can't necessarily have photosynthetic organisms existing in the upper surface waters of those environments.

Um, and so most of the nutrients that go into that environment, uh, what we refer to as a lot through s,

um, so, um, carbon, um, and particulate organic carbon can be derived from the surrounding.

So for example, from overhanging trees that might, uh, drop their leaves into that environment.

Um, and so then we've got that from this carbon, um, coming into the system.

It can be, uh, degraded into smaller subunits,

so invertebrates might break it down into particulate organic carbon, uh, which can be fed on by other organisms.

Um, or it might be, um, broken down into dissolved organic carbon.

So single carbon molecules, um, which can then be fed on by microorganisms like fungi, um, and other bacteria.

Uh, yeah. Um, and so here, of course, we have biofilms as being, um, one of the dominant forms of microbes.

So they're not able to exist in a stable environment when they're up in that fast flowing water.

Um, but they can be protected in biofilms.

And so that's often why we see those slimy rocks, for example, in, um, classifying waters.

Um. Larger bodies of water.

Um, so lakes, um, large rivers. Um, we might refer to as landing systems.

Um, and so these, uh.

Um, not that we we do have the opportunity for stratification, so we can still have phytoplankton, uh, existing stable surface waters.

Um, and they can be responsible for most of the carbon fixation.

So most of the primary production, um, but of course, there is opportunity for plants to also,

um, exist in those environments and contribute fixed carbon.

Um, water is very important. Um, so at the end of this lecture, um, we touched briefly on, um, how we can, um, make sure that water is clean,

um, and how we can monitor it, uh, so that it can be used by us for drinking and for, uh, um, recreation.

Um. We talked about the types of nasty microorganisms that we might not want to have in, uh, drinking water.

Um, so some of those, uh, listed there and they come from all different, um, groups.

Uh, and water regulations. So, uh, we've got this extremely lengthy book, um, which dictates the, um,

the rules around the numbers of microbes that are able to be, uh, found within our drinking water.

Um, for it to be considered safe for consumption. Um, and in a lot of the cases, it's very low.

Uh, we did also talk about indicator organisms.

So for example, um, probably forms, um, which can exist, um, quite stably in, um, water and potentially outlast, uh, potential pathogens.

So if we find those, then we may also find, uh, pathogens.

Um, and then finally, um, in like two, eight, um, we spoke about microbes in terrestrial environments, um, so particularly within soil.

Um, so soil is a great micro habitat for microorganisms, and microorganisms are hugely abundant in soil.

Um, so we can have, um, yeah, billions of cells per gram of soil.

Um, so within the soil we've got soil particles, um, which sort of pack together.

Um, and then, uh, in between those, there's, uh, pore spaces, which are important for the movement of water and for gases.

Um, and that's basically where we would find, um, microorganisms living.

Um, there's lots of organic matter in the soil, so, um, can be dead material, um, decaying.

It can be, um, secreted from plant roots, for example.

Um, so huge amounts of organic carbon in soil, which is why it's so densely packed with microbes.

Um, and we see all different types of microbes living within, um, those environments.

Um in the soil. Environment. Most primary production is from plants.

Um, so we've got our plant residues being fed on by, uh, different groups of microorganisms.

Um, and then, um, those microorganisms can be fed on by large organisms within that environment so the carbon can move up, um, trophic levels.

Um, dead plants is a major part of, uh, organic carbon that we might find in soils.

Um, some of these, um, is not all.

Some of the material in dead plants is not particularly accessible to microbes.

Um, so there will be some amount of easily, uh, consumed carbohydrates.

Uh, but we've got more complex carbohydrates.

So things like cellulose, uh, which are important in plants structurally, um, they can only be degraded by certain types of microbes.

Um, so different fungi that produce secreted enzymes to degrade those into smaller subunits that can be consumed.

Um, and they are extremely, um, resistant materials.

Um, so for example, lignin, um, but it takes very special microbes to degrade.

Um. So these organisms.

Um. Uh, heterotrophs. Um. And they respire.

And so when we disturb soils. So, for example, when we kill soil, um, we can, uh, potentially introduce more oxygen, um,

and increase the opportunity for those types of microbes to degrade that organic carbon.

Um, and it can be lost from soils. And so it can change the health of our soil.

Uh, we can also impact the amount of carbon in the soil, for example, through irrigation, um, which can potentially have the opposite effect.

So, uh, by pouring lots of water onto soil, uh, we're reducing the amount of oxygen in the air for carbon degradation.

Um, plant microbe interactions are extremely important for the health of plants.

Um, I won't go into that slide too much. Um, mycorrhizal fungi?

Um, uh, um, the type of fungi that form intimate interactions, uh, with most land plants.

Um, basically, they are able to deliver, uh, nutrients, uh, from a broader environment into the plant,

um, allow the plants access those nutrients from a broader space.

Um, and they can in return get, um, carbon from the plant.

Uh, we've already spoken a little bit about nitrogen fixation and rose obeah.

Um, and of course, I grew a bacterium as well. Um, so, uh, uh, we'll stop there for module one.

Um, it's. Anyone got any questions around that?

And it was. 73 slides in the short time span.

Sorry about that. Nope.

Uh, so we've got. We should probably move on to module three.

Yeah. Uh. I could do if the zoom link stays open, and I'm happy to continue on past as week gets recorded.

Um, yeah. I'm not sure what happens when it hits 6:00.

It might stay open. I'm not sure. Um, I can probably go fairly quickly over the first half.

Um. So, uh.

Let me just bring that up. Okay. So.

Basically the to half. The module that I did was all about, uh, viruses, um, and went over what a viruses that it's, uh.

None. It's a it's it's an obligate pathogen basically.

So it can't replicate itself once it's outside a host cell.

And so you can, uh, you can debate as to whether they are living organisms or not.

Um. And we went over some of the definitions of what's a variance of variance is the complete virus particle.

And pretty much all viruses have the same core structure that they've got their genetic material, whether it be DNA or RNA,

single stranded, double stranded, encapsulated in a protective protein coat called the um capsid.

And then they can have other layers on top of that. And their genetic material is at least one molecule.

Usually it's one. But we have seen examples, for example, flu influenza virus which has more than one molecule.

And it can be the DNA or RNA and usually not both.

And they can have additional layer. So some of them have a membrane outside that.

And so that you can have single stranded DNA, double stranded DNA, single stranded RNA or double stranded RNA.

And those are examples of ones which would have those. We went over the ways in which you can say how many viral particles you have.

So there were direct measurements and indirect measurements.

Um, so you can directly count, um, what looks like virus particles using say, electron microscope.

Or you can have indirect counts. So things like plaque and size.

Um, and those are actually measuring not the total number of viral particles, but the total number of active viral particles.

So there's a subtle distinction. And we went over a few examples.

So, uh, an example of a double stranded DNA virus that infects bacteria is T4.

And we went over the life cycle. Um, and how, uh, you have this temporal control of, uh, transcription.

And so you have early genes which are involved in the initial takeover,

and then you start producing genes involved in replicating, uh, the, uh, viral DNA.

And then later on, you start expressing new packages for the virus,

and then eventually you express what lives as the cell and goes and releases the assembled viral particles.

Receive call that will temporal control. And we compared that to a eukaryotic virus.

So in this case herpes virus. So that comes into a cell.

It's a membrane bound uh viral particle.

So the nucleocapsid comes into the cell. And then that transported to the nucleus along microtubules.

And then the DNA comes into the nucleus. And again you get that temporal control.

So the immediate, um, uh, transcription allows takeover of the cell.

And then you get the next set of um transcripts which allow for replication.

And again, the replication code goes through these concatenates.

And then you get the very light transcripts which produce new viral particles.

Those go out to get encapsulated in a membrane and then go out and infect expanding cells.

So a lot of similarities in the temporal control of, uh, the this DNA virus from bacteria and one from humans.

Um, and then we looked at the other sort of life cycle that phages can, um, viruses can go through, which is um.

Basically existing in a non lytic, uh, way with a host cell.

So the classic example of this is something like bacteriophage lambda which is a temperate phage,

integrates itself into the host chromosome and is then replicated passively.

And then we went over some of that evolutionary arms race that you can see between phage and their hosts.

So I won't go into this in great detail, but you've got things like restriction enzymes which cut the DNA, um, and all kinds of other, um.

Methods that phage used to get round this, for example, um, using hydroxy methyl cytosine in their DNA and then glucose creating that.

So adding a glucose molecule on to that to avoid being recognised um by the host restriction enzymes.

And then you've also got things like the Crispr system.

Um, and we went over it. So you've got genes for incorporating new repeats into this array.

And then each of those repeats encodes a specific RNA molecule which can then be used to look for homology.

And so the RNA gets loaded into this enzyme called Cas9.

And you've got what's called this guide RNA. And so that short segment of that RNA looks for homology in invading DNA.

And if you find perfect matches and you've got an associated sequence next door to it called the Pam site, which is usually just angry,

then if you've got both of those things, then you cut both strands of that DNA and then you've killed the virus.

And so that's how Crispr works. And then we went over, um, how enveloped viruses get into the cell.

So, um, in a lot of cases, you've got some specific recognition between, say,

a spike protein and a cell surface marker, and that can lead to membrane fusion.

And then that delivers your nucleocapsid into the cell, and then your DNA or RNA can come out.

And so an example of that that we went over would be something like corona virus.

You also have underside ptosis sometimes viruses.

So rather than membrane fusion the whole thing can get taken inside the cell.

And then you get an acidified end design which then leads to fusion inside the cell of this membrane,

with the vacuole leading to release of the nucleocapsid in the cell.

You can also find in eukaryotic virus these ones which actually directly to the liver straight into the cell.

So they would um get under site host of the nucleocapsid.

And then the nucleocapsid injects its DNA or RNA into the cell.

And then again very quickly going over this. You can have the different forms of that genetic material.

It can be an RNA and it can be either the positive or negative strand of the omni.

So remember that a positive strand RNA is effectively the same strand as an RNA.

So that could be read to translate protein in terms of replicating this.

You need an enzyme which can copy RNA into RNA.

And so that's an RNA dependent RNA polymerase.

And so that would initially take your RNA copy and copy the complementary strand which is the negative strand.

And then you can use that negative strand to copy multiple positive strands.

And so those positive strands then get packaged into the new virion.

And again an example of a positive RNA virus.

Are things like the corona virus that we went over and we went over again, some of the corona virus, um, life cycle,

how it produces these two poly proteins, which then get protein list to give you what are called non-structural proteins.

And there are between 13 and 18 of those which go and replicate the RNA.

And then you've got these other subcu, gnomic, uh, mRNAs which are produced, which then produce the capsid later on.

And then you get assembly of this, um, in the endoplasmic reticulum,

assembly of a new viral particle, which then gets released, you know, outside the cell.

Um. So you can also then have double stranded RNA viruses.

These are quite rare, but there are examples of them things like the rotor viruses.

Um, and they again can they have both a positive and negative strand.

They can be peeled apart and uh, used to produce mRNAs and then replicated to produce more positive and negative stranded RNA.

And then you've got your negative strand nominees. So they come into the cell and they are the opposite strand to an RNA.

And so to get an RNA you need an RNA dependent RNA polymerase to read that information and produce the RNA form.

You can then have complete copies, which are the positive strand, which can then be copies completely again to form the complete negative strand.

And so you've got a replicative form and then you've got an m RNA form.

And a really good example of this is something like the influenza virus.

So it comes in with a negative strand RNA. And sometimes that entire thing is copied.

And that gives you a positive strand RNA which can then be copied to make more negative copies.

But in order to produce proteins, it then needs to do something else to get the positive strand m RNA.

It does something different, so it doesn't encode its own enzymes to produce a cap.

So you carry otic. Maharani needs a couple of distinctive things that mark it out as an MRI.

It needs a five prime camp and a three prime. A tail coronavirus encodes the enzymes, its own enzymes to produce those.

Influenza virus has to steal them. And so what it does is it takes the cap.

So this is a cap. So it's a five prime to five prime linkage at the very end.

Um and what it does. So this is the flu polymerase is it's got an and a nuclease.

So it recognises uh a transcribing RNA polymerase to within a cell.

It, then latches onto it, waits until RNA has come out and been capped,

and then it flips out that nuclease and cuts off that five prime cap and about 12 nucleotides of the RNA.

It then flips that RNA back inside its own active site, and then uses that as a primer to then synthesise RNA opposite its own genome.

And so that's what caps matching is.

So it's really important um, a that adaptation that the influenza virus has got around this problem of m RNA needs to be the positive strand,

but they need to be marked out as mom and niece by having this cap on one end and a poly a tail on the other end.

And then we went over the different sort of flavours of the coronavirus,

vaccines that are made and the relative advantages and disadvantages of each of them.

So that's a very quick run through that. So if anybody's got any questions, feel free to ask.

Nothing's coming up. Um, I will stop there, but, uh, I will stay on for a little while.

So if you do have any questions, feel free to put them in the chat and I can answer them just in the chat.

While Carl goes on about the second half of module three.

Yeah. Okay. To start. Okay.

So in the second. Were there any questions?

No.

Um, second part of module three, uh, was basically, um, looking a little bit into applied microbiology, um, and how we can use microbes industrially.

Uh, in particular. Uh, but the first lecture was devoted to genomics and functional genomics.

Um, which is one of the ways that we can get huge levels of information about micro organisms.

Um, from relatively now, routine experiments.

Um. Uh. Um.

There we go. Good for you. Pop ups. Uh, so genomics and functional genomics, um, centres largely on sequencing technologies.

Um, and so, uh, during lecture uh, five in module three went through a few different sequencing approaches that have been developed.

Um, and they've, um, rapidly, uh, evolved over the past 20 years.

Um, so back in the 1970s, um, Sanger and his colleagues, uh, developed the Sanger sequencing method,

um, using terminator, uh, nucleotides, uh, to sequence, uh, DNA.

Uh, that was, uh, subsequently automated, uh, into, um, uh, a more high throughput process.

Uh, but since then, uh, we have now, um, what we call next generation sequencing technologies.

Um, so two of the major types of sequencing that we use these days, uh, um,

Illumina sequencing or sequencing by synthesis, uh, and Oxford Nanopore sequencing.

Um, so Sanger sequencing is based on pcrs, uh, but we incorporate terminated nucleotides at different points,

or they get incorporated randomly during that PCR, um, and create a single stranded, um, uh, piece of DNA of a particular length.

Um, so depending on where that terminator nucleotide came along.

Um, and then we can use electrophoresis to separate out all of those single stranded, um,

pieces of DNA and basically read off the sequence based on the terminator that's present at the end of that single stranded piece of DNA.

Um, sequencing by synthesis, um, uses PCR steps.

Uh, the PCR occurs on a flow cell. Um, and you end up with a little cluster of, uh, DNA, um, strands that are all of the same sequence.

Um, and then the, uh, so you can think about synthesis steps where the different nucleotides have flowed into the flow cell, um, in different stages.

Um, and each of those has, uh, a different, uh, fluorophore attached to it.

Um, and you can see which nucleotides present, uh, next in the chain of DNA based on the fluorescence of the spot,

um, around a little cluster of DNA on the flow cell.

Um, during each cycle of that, um, sequencing by synthesis.

Um, and then more recently, we've gotten, um, very fancy technologies.

Um, so Oxford Nanopore, um, uses little, um, nanopores, as the name suggests, a little proteins.

Um, that strands of DNA or potentially RNA are pulled through.

Um, and the resistance across the force of the electrical resistance across the pool, um,

is indicative of the nucleotide that's present within the pool at that point in time.

Um, so we can sequence, uh, DNA in this way. Um, and we can get very long sequence reads, um, doing that.

Um, so in the past, we might have used, um, DNA sequencing just to look at, um, a PCR product or a small piece of DNA.

Um, but now, uh, we can basically, um, reassemble whole genomes.

So with those different sequencing technologies, um, we can get sequence rates.

Um. Uh, and those sequence reads will have overlapping, um, pieces of sequence,

uh, that we can then use, um, to put the genome back together, so to speak.

So stitch it back together using bond phonetics based on overlapping sequence rates.

Um, this is, as you might imagine, much easier with long sequence reads.

Uh, because the region of overlap is much longer. Um, and so that's why technologies like Oxford Nanopore, which give us long sequence, reads, uh,

potentially more useful for this type of sequence, um, in, uh, regenerating, um, whole genome sequences.

Uh, once we've got that genome sequence, it can tell us a lot about the organism that it came from.

Um, something that we'll do is annotate the genome.

So basically try and identify parts of or bits of sequence that encode for genes.

Um, so we can do this by looking for start and stop curtains and finding open reading frames that don't have any stop curtains.

And within a lot of those open reading frames will have a coding chain.

Um, so we can then take those coding sequences, compare them to databases.

We might compare them, um, to, um, a huge database of other known gene sequences using Blast.

Um, we can also do other types of searches looking for conserved domains.

Um, so a lot of, um, sequenced domains, um, or conserved sequence motifs, effectively, uh, associated with particular functions.

And so we can start to infer the function of different genes within the genome.

Um, and from that build up, um, metabolic models, um, about how organisms may make living.

So there's certain genes that are associated with different types of metabolism.

Um, and if we find those within a genome, um, it'll give us information about how that organisms is able to,

um, get its organic carbon, how it's able to get, um, energy and so on.

Uh, once we've got whole genome sequences as well, uh, we can compare, um, different organisms.

Um, so if we've got two strains of the same species, but we know that one is better at causing infections, um, we can potentially,

for example, compare the genomes of those two organisms, um, and potentially identify novel virulence factors.

Uh, he was talking about virulence factors earlier on. Um, these are things that help bacteria to infect.

Um, we can identify information about those by comparing organisms that, uh, better and less able to infect, for example.

Uh, we can also use sequencing to access information about organisms that we haven't been able to isolate.

Um, so I mentioned metagenomic sequencing earlier. So here we go out to environment, um, and take a sample.

Isolate all the DNA from that sample and sequence it.

Um, again, we need to do some, um, type of, um, assembly, um, or classification of the sequence rate that we get from how they could approach.

Um, sometimes this is referred to as binning.

So we'll try and find sequences that have similar characteristics that they might have a similar GC content,

for example, um, or what we call a different camer content.

So the um, the ratios of different um, strings of sequences might be similar between different reads.

Um, that allows us to put them into groups.

Um, and those different groups probably correspond to different, uh, organisms within that um, sample that we originally extracted.

Uh, we can then do things like taxonomic profiling.

So we saw the Tree of Life made by Gillian Banfield, which is based on these types of sequences, um, which have just come from natural environments.

Uh, again, if we've got, um, samples from an infection, um, we can start to look for virulence factors.

Um, they might be associated with different taxonomic groups.

And so we can start to learn things about the types of organisms that are present in the environment,

um, and might be causing the phenotypes that we're interested in.

Um, and we can also do functional profiling.

Um, so again, using blast searches and domain searches to try and determine what types of genes are present in that environment.

Um, to get an idea about the functions that, uh, present in that environment.

Um, and they had the organisms there, uh, making a living.

Um, sometimes genomics alone is not enough to give us the information that we need.

Um, and so then we'll use functional genomics, uh, potentially functional metagenomics.

Um, so this is how the genes, uh, used, uh, within the organism, um, to ultimately, um, get to a phenotype.

Um, so we can look at the level of transcripts using transcriptomics so we can sequence RNA from organisms.

Um, we might treat an organism, um, with, uh, for example, an antibiotic, and see how it responds to that antibiotic.

Um, and genes that are induced.

So they're more highly expressed in the presence of that antibiotic may be involved in conferring resistance to that antibiotic.

Um, and it's that stimulus, um, there's additional levels of information about how microbes respond to their environment,

um, rather than just which organisms are able to exist in a particular environment.

Um, proteomics. Um, basically we can get similar information, but he at the protein level, um, proteins are typically longer lived than transcripts.

So there are different situations where you might want to use one or the other technology.

Um, depending on, um, type of experiment that you're doing.

Um, metabolomics can tell us, um, uh, about the metabolites within an organism.

Um, lipid omics and block dynamics are basically, um, related to metabolomics.

Um, and this can tell you, for example, um, if we're interested in, um,

the degradation of an environmental pollutant, um, what types of products is that pollutant being degraded into?

So what metabolites are present when we expose an organism to that pollutant?

Oops. Um, we're also able to do now what we refer to as final mix.

Um, so we can, um, take an organism, expose it to, uh, an array of different environmental conditions.

So for example, uh, grow it in different carbon sources and see which of those carbon sources it's able to, to consume.

Uh, to get additional information about the organism. Um, and so that might be important when we're trying to um,

and we'll look at this in a few slides time, I guess, um, trying to identify a good by a catalyst,

um, which can degrade a huge range of different carbon sources, um, from, uh, a waste product and convert it into something useful.

Um, and we can also do techniques like chip seek.

Um, so here we can see where regulatory proteins abound within the genome at different points in time.

Um, so to give us information about how genes are regulated in response to different environmental pressures.

Um, so related to transcriptomics, we can, um, link those types of information.

Um, and so this gives us, um, a huge array of additional information.

Um, which might potentially be useful if we want to engineer an organism to do something for us.

Um, and so with that in mind, we then went on to look at a little bit about, um, my, uh, industrial microbiology.

Um, and so we can use microbes to make things like the spider silk proteins that are present in this tie in this dress,

um, made by this company, bolt threads. Um, so potentially renewable.

Um, textiles. Um, we might need to engineer an organism.

Um, and I won't speak about this for too long because it's stuff that we have covered before.

Uh, there are different ways that we can engineer microbes, uh, introducing DNA into an organism, for example,

for new enzymatic reactions that might not be present in that organism, uh, but, uh, required to produce, um, something that we want.

So, for example, in the case of these spider silk proteins, um, the bacteria, um,

and I think also yeast that have been engineered to make those, um, thread like proteins would naturally do that.

They'd have no need to make those spider silk proteins. So the genes to do that were introduced into those organisms, um,

so that they could be expressed recombinant like in those organisms to, to isolate large volumes of that, um, material.

Uh, so restriction enzymes. Electrophoresis, cloning, cloning vectors PCR.

Um, a things that you've probably all heard about before.

Uh, we can take DNA from an organism, chop it up with the restriction enzymes, turn it into a plasmid or a cloning vector,

um, and then introduce that into a new host, uh, more sophisticated approaches.

Uh, we now have, based on, um, Cas9 genome editing, um, and other types of synthetic biology.

Uh, so I won't talk about cloning vectors.

We've got plasmids, bacteriophages, viruses, cosmonauts, artificial chromosomes, the different vehicles for introducing DNA.

Um, we can introduce it through transformation.

So some bacteria, um, will take up DNA naturally from their environment and incorporate it into their genome.

Uh, we can also do this, um, chemically or electrically.

Um, so introduce plasmids, for example, um, conjugation where there's interaction between uh,

donor and incipient um, can be used to transfer uh, um, conjugated plasmids from one strain to another.

Um, and transduction. Um, we can take advantage of those little phage that, um, he was talking about, uh, to introduce DNA into organisms as well.

Uh, he also spoke about, um, Crispr cas, um, as a mechanism of, um, viral, uh, resistance or as a viral defence mechanism.

Um, this is also, of course, been taken advantage of, uh, in biotechnology, uh, to introduce, uh, mutations directly into the genome,

so directly into the chromosome, um, which is often not trivial to do in most organisms that we work with.

Um, so. The Cas9 um, using a guide RNA that we design can cut, uh, DNA, uh, targets that, uh, we can get,

um, mutations introduced through that process, um, through non-homologous in joining.

Um, so potentially inactivate genes in that way.

Um, or we can introduce new pieces of DNA.

So if we put in, um, donor DNA, we can get recombination between where, uh, the DNA was cut, um, and our donor DNA, um, to repair that damage.

Um, but in the process, introduce pieces of DNA that we want to have in that genome.

Um, so a very useful tool that is being constantly developed, um, and using these approaches in industrial microbiology, um,

it's very often the case that you might need to do several iterations of creating your, um, target by a catalyst, um, to get an optimised design.

Um, so you'll often go through these design build and test phases.

So you make a hypothesis potentially based on your genomics or functional genomics,

about how you could engineer your organism to be more productive, um, in your design phase.

Then you'll go away and use the tools that we've got to to build that and then test it.

Um, and if it, um, has, uh, a good effect, you might, um, take it and start to use it industrially.

Um, but to get to a level that you need to be commercially viable, you may need to go through this cycle.

Um, several times. Um, to make things a little bit more efficient.

Um, we now have, um, various tools in synthetic biology.

Um, so we can create new genes, or we can get genes from organisms, um, that might not have been isolated.

So from metagenomic sequences. Um, we can synthesise them chemically and then introduce them into organisms,

um, and test their functions, um, and use them in, um, genome engineering.

Uh, we can also use synthetic biology, um, to introduce, uh, a large number of mutations into the gene that encode for enzymes.

Um, so we can make predictions. And people are getting reasonably good at this using, um,

AI and machine learning to try and predict what mutations could be introduced into enzymes to improve their function.

Um, once we've done that, we can then go and have that new gene synthesised.

Um, and using synthetic biology, introduce it into our target organism.

Um, and I mentioned previously the mycoplasma um, as being completely synthesised, having their genomes completely synthesised.

Um, this is now being done with multiple organisms.

So, uh, E.coli, which is our laboratory workhorse, has had its genome completely received, resized.

Um, and also several chromosomes in Saccharomyces cerevisiae have been completely chemically synthesised, um, and put back into the organism.

Um, this is useful because you can strip out potentially genes that, uh,

needed in that organism to, to use it as a catalyst for whatever it is that you want to produce.

Um, and you can do that in an efficient way. So it would take a long time to delete hundreds of genes from genome.

Um, but you can do it, um, in one step, potentially, uh, with this type of, uh,

synthetic biology and to increase the efficiency of your organism by stripping out things that don't need to be in there.

Uh. Lecture seven went on to talk about industrial microbiology in more detail.

Um, so starting by looking at microbial bio catalysts, um, and your initial choice of an organism.

Um, so most organisms used in industrial microbiology come from natural materials so isolated from the environment.

Um, and as we looked at in the early lectures, there's huge diversity in the tree of life that we haven't tapped into yet.

So there's potentially organisms out there that can do all sorts of different, um, catalytic processes.

Um, and we just need to go and look and isolate them, look for them and isolate them.

Um, sometimes you might want to use a consortia of microbes.

Um, so they might be cross-breeding between um, of metabolites, between different organisms, um, to get to an end product that you want.

Um, so one organism alone might not be able to do that. Um, you might do, uh, succession.

So you have one organism doing one conversion taking um, uh,

metabolite which might come from some sort of waste product, um, and, um, converting it into something else.

Um, and then a second group of microbes coming along and modifying that further.

Uh, you also need to consider preservation, uh, in choosing, um, by catalysts,

because you want to be able to preserve your organism without it changing, um, or being mutated randomly over time.

Uh, and of course, you might need to engineer your organism.

And this can be done by, um, targeted approaches, which we looked at, um, a few minutes ago.

Um, or you can potentially do it through, um, natural mutation.

So just, um, growing your organism across generations and allowing mutations to be introduced that you might be able to select for, uh,

or you could speed that process up with, uh, a mutagen, um,

and then select ideally for the organisms that, uh, most fit, um, and able to do your process.

Um, the easiest.

Um, so, for example, if you've got a particular feedstock, um, you might be feeding your microbe, um, a waste product you can quite easily select for,

uh, mutants that are better able to use that feedstock because they'll be growing faster and outcompete, uh, the others in that population.

Um, and so over time, through multiple generations,

you're able to develop a better by a catalyst that can consume that feedstock that you're feeding the cells.

Um, it can be a little bit harder to select the organisms that are producing something that you want.

Um, because often that has a negative fitness impact.

If they're producing a high amount of, for example, a metabolite that you might need, um, for an industrial process,

um, once you've got your organism, um, we need to be able to scale up and industrial scale culture.

Um, this ideally, again, we'll use, um, a cheap feedstock.

So this might be waste products, um, from agricultural or forestry residues.

Um, and Ian can probably talk about that for a while from his work with ethanol technologies.

Um, using those types of, um, waste products, uh, to feed to yeast.

Um, to get them to produce ethanol. Um, also, uh, meat processing residues could provide, um, additional nutrients, um, for the growth of microbes.

Um, crude chemicals, which are cheap. Um, it would be good to be able to recycle the culture medium.

So any, uh, metabolites remaining could be put back in.

Um, and ideally, uh, moving forward. Um, minimal sterilisation.

So using microbes that can outcompete others, um, can potentially increase the efficiency and reduce costs.

Uh, in industrial scale culture.

Uh, typically, the growth of microbes at an industrial level requires, um, quite precise control of temperature, oxygenation and so on.

And so these types of stone fermenters, um, can be used to keep cells in suspension.

Um, we can have new nutrients coming in, uh, potentially, um, products coming out across time,

um, maintaining a very stable environment in those, um, fermenters.

Uh, but of course, there are many alternative types of industrial fermentation, um,

including solid state corrections that are necessary for producing some types of microbes, uh, some types of products.

Uh, there are a huge array of different products that we can get from microbes.

Um, other engineered or natural. Um, so some of these, uh, primary metabolites.

So things like amino acids and nucleotides, um, lipids, carbohydrates, um, fermentation and products, uh, a lot of these are used, uh, in food.

Others are used as fuels. Um, also enzymes and other proteins, uh, a lot of enzymes that we use in washing detergents, for example,

uh, produced uh, in industrial microbiology, um, other proteins, uh, maybe used for vaccines, for example.

Uh, so we can also use microbes to produce those different components.

Um, and then distinct from primary metabolites, uh secondary or specialised metabolites.

Uh, and we spoke about some of these uh, in relation to the acting of bacteria, um, uh, organisms like streptomycin, uh,

which produce a huge array of bioactive metabolites, which they typically, um,

export into the environment to elicit some sort of response in their environment.

Um, so classic example is antibiotics.

And these bacteria probably produce these to compete with their neighbours in the highly competitive soil environment.

Um, but also other types of um specialised metabolites produced um, which have other biological effects.

Uh, so this gives a few specific examples of different products in industrial microbiology, um, and the organisms that can produce them.

Um, I won't go through them in detail, but it might be good to know a couple of these.

Um, and biopolymers. So coming back to the levelling dress, um, that I showed in the first slide, I think Carla Zampatti dress, um,

made from spider silk proteins, um, a type of biopolymer that has been produced, uh, in yeast and in bacteria.

Um, here we can see similar silk proteins being used, um, in this mascara, um, to make, uh, eyelashes more voluminous.

Uh, other types of, uh, biopolymers are also, um, produced for different, uh, industrial applications.

Uh, and then finally, uh, the last lecture, uh, in this series, um, talked about the use of microbes in biodegradation and bioremediation.

So basically how we can use microbes to clean up our mess.

Um, so one of the classic examples of that is the use of microbes in wastewater treatment.

Um, so the, uh, several processes that occur in wastewater treatment.

Uh, there's a primary treatment where we've got, uh, physical movement, uh, removal of solid materials.

Um, so basically through flock formation.

Um, and so you can see, uh, that here.

And so we have, uh, flux settling out, um, and we end up with this clear liquid, uh, which, um, is still full of organic nutrients,

uh, dissolved organic, um, carbon and other nutrients that we need to remove before we can put that back into the environment.

Because if that gets pumped straight back out into the environment, uh, we can have environmental damage, um, through eutrophication and so on.

So we need to, um, process that. Um, and a lot of that processing is done, uh, with microbes.

Um, so we've got microbial growth, um, converting that dissolved organic matter, uh, into microbial biomass, um, and potentially carbon dioxide.

Uh, one of the ways that that can be done is with these trickling type ponds.

Um, so this, um, primary treated wastewater is trickled down over, uh, um,

some sort of substrate, uh, potentially rocks which have microbes growing on them as a biofilm.

And along the way, um, that dissolved organic matter gets, um,

consumed by those microbes and converted to that, uh, microbial biomass and that biomass.

Then, uh, we can refer to as sludge, uh, that can go into anaerobic digesters, um, archaea and other organisms in their processing that,

uh, producing methane, which is a potential, um, source of energy so it can be burned for energy.

Um, and then there can be additional, uh, tertiary treatments where we remove things like volcanic nitrogen.

Um, so via the nitrogen cycle, uh, nitrification, denaturation, uh, or using animals,

uh, bacteria, which are able to convert that organic nitrogen back into nitrogen gas.

Uh, microbes can also be used in bioremediation.

Um, so here we've got degradation. Um, and there are different types of biodegradation that can be minor changes.

Um. Um, so for example here we've got this molecule two for diclofenac acid, which is a herbicide, um, that can be chlorinated.

Um, so basically just have the chlorine, uh, removed, you know, minor change.

Um, and then this molecule would be more prone to degradation by other microbes.

Uh, we can have fragmentation. So here that same molecule is converted into two four dichloromethane and acetic acid.

Um, some of these breakdown products may be more toxic than the original product.

So we do need to be careful. Um, if those types of, um, conversion processes are occurring.

Um, but the gold standard in bio remediation is basically complete mineralisation.

So we take, uh, um, uh, harmful molecule in the environment and microbes can, uh, degrade that fully down to carbon dioxide.

Um, and it's, um, elements. Um, some compounds that are recalcitrant to biodegradation.

Um, this type of chlorination or um, more generally, elimination, um, can make molecules, um, less prone to biodegradation.

So there's far fewer enzymes that can act on those types of molecules.

Um, and so far fewer microbes in the environment that can degrade them.

Um, and we do this because we want these chemicals to persist in any environment.

So there's not much point in spraying herbicide if it's going to be degraded by microbes within a day or two.

You want it to exist in that environment for long enough to have the effect that you want it to.

Um, but then ultimately it can become a pollutant if it's used at to high concentration.

Uh, one way that we can encourage, um, the growth of natural communities to degrade things is to slightly modify the environment.

Um, so, for example, um, hydrocarbons, um, are best broken down through, um, oxygenic, um, enzymatic pathways.

So they require oxygen.

Um, and in this little figure here we've got an example where we've got, um, spill of hydrocarbons, um, down the bottom of this, um, chemical plant.

Um, there are microbes down there that can degrade them. Um, but to sort of increase their activity, uh, because it's so far down in the ground,

we need to pump in some oxygen to have them, uh, more efficiently degrade that chemical.

Um, we can potentially put in nutrients.

Um, so they might be a lot of carbon in our target chemical, but not much nitrogen or other elements or organic forms of those.

So they could potentially be added, uh, to, um, to improve degradation efficiency.

Um, bio augmentation, um, is where we're adding microbes to an environment to try and degrade a pollutant.

Um, these microbes might be naturally occurring in that environment.

So we may have taken some material away, enriched organisms that can, uh, degrade.

Uh, that pollutant and put them back at higher numbers. Um, or they might be modified strains.

Uh, but we do need to be mindful of regulations.

Um, in introducing modified strains into natural environments.

So, um. If we've, uh, generated those strains through things like, um, assisted laboratory evolution, um,

then they're more likely to be considered, not genetically modified so they can be put into an environment.

Um, but if we've made targeted genetic modifications, it's less likely that regulation will allow the release.

Um, at least in, um, a short time frame.

Uh, and we can also use microbes to work with plants, um, to try and, um, remediate environments.

Um, so in an earlier lecture, we talked about plant microbe interactions very briefly.

Um, microbes can promote plant growth, um, and improve their tolerance to stresses.

Um, they can potentially also influence the uptake of potential pollutants.

So for example, um, heavy metals, um, we do see some plants that can take up huge concentrations of heavy metals.

Um, so for example, these trees, uh, which are found in Yuma, um, which has high concentrations of nickel in the soil,

uh, have green sap because they're able to extract so much nickel from the soil.

Um, and so we can potentially use microbes to enhance these processes, um,

and extract things like heavy metals, um, which, of course, can't be degraded.

Um, we can extract them in a more efficient way from the soil, um, rather than just dig up the soil and move it somewhere else.

Um, which is the current approach to remediation in a lot of cases.

Um, and finally, uh, we can also use microbes, um, to help us in bio remediation, um,

and also to prevent, uh, the use of chemicals that might need to be cleaned up in the future.

Um, so, for example, uh, microbes can be engineered to detect microbes in and,

uh, detect harmful chemicals in the environment, like pesticides and herbicides.

Um, using biosensors. Um, so a lot of, uh, regulatory proteins, for example, in, um, bacteria will bind to,

um, some of these compounds and elicit, uh, gene expression changes.

Um, we can link those, uh, regulatory proteins to fluorescent proteins, um,

to create a little biosensor for those compounds so we can detect concentrations of those compounds, um, in a fairly simple and cheap way.

Um, and we can also use microbes as alternative to alternatives to, uh, things like pesticides.

Um. Largely through their production of specialised metabolites again.

Um, so a lot of microbes that colonise plant surfaces will produce, um, specialised metabolites, um,

largely because they want to compete with other organisms and they want to exclude them from that environment.

So they will try and inhibit their growth with these antimicrobials, antifungals, antibacterials, um, and they can protect plants from disease.

So you can see an example of that down here. Um, um, so we've got a control plant with a nice well-developed root structure.

Um, here is a plant that's been, um, infected with, um, an organism called PM ultimate.

Um, and that has affected root development. Uh, and here is a plant that's been infected with the same, um, pathogen, uh, but in the presence of, uh,

beneficial bacteria, um, and it's protected the plant from the, um, pathogen that the roots of develop normally.

Um through these specialised metabolites, um, in an organism that I've worked on a bit in the past,

um, which is the strain or the genome of a strain of Pseudomonas.

Um, it also produces insecticidal toxin, um, which is called Gm-csf because it has the activity of making caterpillars floppy.

Um, because of its, um, insecticidal activity.

Uh, and that brings us to the end of the revision lecture.

Hopefully we're still recording and everyone's still online.

Um, does anyone got any question? Thanks for staying on.

Um, just while we wait to see if there are any questions.

Just to remind you. Um, the exam is going to be two hours.

There's ten minutes of reading time and split into three sections.

Six questions in each section of which you have to do four.

So you have 2 to 12 questions out of 18 in total.

So you can use that ten minutes reading time to look through.

Decide which of those questions you're going to do. And then you'll have two hours to complete the exam.

Good luck to you all. Um, hope you do really well and yeah.

Hope you enjoyed the course. And if you haven't already done the, uh, feedback on the course, we would love to hear your feedback.

So please to fill out that, um, survey.

Okay. I'm not seeing any questions. So, um.

I'm guessing that's it. Thank you all.

I will stop recording and. Wish you good luck again.

Yeah. Good luck everyone. Thanks.