BIOL0510: Microbial Biotechnology

What is the biotechnology timeline?

• pre-Pasteur (fermenting drinks/food)

• Pasteur (industrial fermentation and organic acids)

• antibiotics

• post-antibiotics (producing amino acids, enzymes, etc)

• synthetic microbio (vaccines, insulin, biofuels)

What is microbial biotechnology?

the use of microorganisms, cells, or cell components to make a product (ex. foods, vitamins, enzymes)

What is erythropioetin?

a protein/hormone that encourages the production of red blood cells

What is a vector?

a DNA molecule used as a vehicle to transfer genetic material (ex. a plasmid or virus)

What are shuttle vs expression vectors?

shuttle: usually plasmids that can propagate (spread) and facilitate gene transfer between two or more species

expression: transfer a gene and express the protein product of the gene

How do you make a shuttle vector?

1. introduce new DNA into existing organisms’ DNA to make recombinant DNA

2. introduce the recombined DNA into an animal to make them transgenic

What is recombinant DNA?

DNA created by combining genetic material from different sources/species to produce a ‘recombined’ genome

What is a transgenic animal?

one whose genome has been altered by the introduction of a foreign gene from another species

Describe the steps of genetic engineering using recombinant DNA technology

1. isolate a vector (ex. plasmid) from a bacterial cell

2. cleave your DNA segment of interest using enzymes

3. insert the gene of interest to the vector

4. introduce the vector to a bacterial cell for amplification

5. clone cells with the gene of interest to either produce copies of the gene or to produce proteins by that gene

What do we use to cut and paste a gene of interest into a plasmid?

restriction endonucleases: DNA cutting enzymes that cut at the ends of your gene of interest

ligases: link together gene fragments

What are the different ends you can make from cutting DNA?

sticky: cut pieces of DNA with an overhang

blunt ends: evenly cut ends

Why do bacteria have restriction endonucleases, aka what are their other functions?

• they are antimicrobial against phages

• they can degrade other bacteria that may infect them with their DNA

(defense strategies)

How do we get a new bacterial cell to take up a plasmid? 2 ways

• transformation: forming pores in bacteria to induce plasmid uptake

  • CaCl2 + heat shock

  • Electroporation

• Transduction: using bacteriophages that contain engineered plasmids to spread the plasmid via infection

How do we make sure that the bacteria received the plasmid? List 3 common selection markers

In a process called selection

• common markers/strategies used to verify selection

  • antibiotics

  • fluorescent marker (GFP)

  • sequence the DNA

Describe the first step of selection used to verify that the correct plasmid (with ampR and LacZ disruption insertions) was uptaken

place transgenic bacteria in a plate with ampicillin; bacteria that took up the plasmid with the ampR insertion will continue to grow/display resistance to ampicillin

Describe the 2nd step of selection used to verify that the correct plasmid (with ampR and LacZ disruption insertions) was uptaken. Define lacZ and xgal, discuss the two possible outcomes, and explain why you would select for white colonies over blue.

lacZ: a gene that makes galactosidase

X gal: galactose with dye on media

outcomes:

1. X gal (on plate) + galactosidase (LacZ is functional) = blue colonies grow

2. X gal (on plate) + no galactosidase (LacZ is NOT functional) = white colonies grow

selection:

• we want to select for and clone white colonies: white colonies are white because they disrupted LacZ activity (which is what we intended for the disruption insertion to do)

• we don’t want to select for blue colonies: blue colonies are blue because the disruption insertion ended up inserting outside of the LacZ gene or not inserting at all

Why do both ampicillin resistance and lacZ screening need to be conducted in this example of gene editing?

• ampicillin selection ensures you’re only working with cells that took up the plasmid

• lacZ screening allows you to distinguish between cells with the original non-recombinant plasmid (blue colonies) and those with the recombinant plasmid (white colonies)

What do we do to the bacteria with recombinant DNA after selection? Define the step and how its conducted

purification: isolating the desired product (often a protein) from bacterial cells

How:

• give them media and let them grow

• Conduct high-performance liquid chromatography: a general process that detects for proteins of a certain size that match the desired protein product

List some drugs that are made via genetic engineering/cloning

insulin, human growth hormone, estrogen, steroids, epinephrine

How did diabetic people get insulin treatment before cloning?

• received insulin injections produced by cow pancreases (14 cow pancreases/1 person/year)

How did people with stunted growth get human growth hormone before cloning? What is the risk associated with that method?

human cadavers; 18 cadavers were needed/person/year; risk: potential transmittance of human pathogens

List 2 pros to protein cloning

• increased safety of supplemental drugs

• come from reliable, affordable, and purified sources

What is gene therapy?

using DNA to treat diseases by

1. replacing defective/missing genes

2. removing genes

How does an adenovirus vaccine (ex. COVID vaccine) work?

1. the gene for a specific antigen is inserted into a non-pathogenic version of the target virus —> creates the vaccine

2. the vaccine is injected into a patient in vivo or a patient’s cells ex vivo

   • the virus part of the vaccine infects the patient’s cells so they can express the antigen on their surfaces

   • the patient’s immune system can now detect the virus and maintain immunity to it upon external exposure

What are the safety issues associated with recombinant DNA treatments/products?

1. avoid accidental release

2. GMO crops must be safe for consumption and for environment

3. lower diversity of plants

4. humans — revertant

5. few are approved

What does it mean for a vaccine to be revertant?

an attenuated virus used for gene therapy becomes pathogenic in the host

How is CRISPR used as a gene editing tool; use sickle cell gene editing as a case study

1. remove a sickle cell patients’s HSPCs

2. genetically modify them ex vivo

3. reintroduce them into the body