Chapter 12: Modern Applications of Microbial Genetics

Replication

Dna to Dna x2

• dna polymerase creates 2 dna molecules from 1

transcription

dna to mRNA

• takes dna to make rna (still has that dna it used as well)

reverse transcription

rna to dna

• only can happen in viruses

• takes rna and uses it as a template to create dna (still has the rna)

• reverse transcriptase does this

translation

mRNA to protein

• takes mRNA and decodes it to create protein (still has that mRNA)

gene expression control

regulating when genes are expressed

• in abscense of lactose, the lac repressor binds to the operator and transcription is blocked

• in the presence of lactose, the lac repressor is released from the operator and transcription proceeds at a slow rate

why genetic engineering matters

• scientists can manipulate biological processes “in-vitro” (in test tubes)

• understanding these processes allows us to hack genetic machinery

• applications: create transgenic organisms with new capabilities

conjugation

transfer of dna through direct contact using a conjugationpilus

transduction

mechanism of horizontal gene transfer in bacteria in which genes are transferred through viral infection

transformation

mechanism of horizontal gene transfer in bacteria in which naked enviromental dna is taken up by a bacterial cell

• the naked enviromental dna gets taken up by a bacterial cell or prokaryote

• this happens spontaneously but weve found ways to do it in the lab

transposition

process whereby dna independently excises from one location in a dna molecule and integrates elsewhere

mechanisms of genetic diversity in prokaryotes

conjugation, transduction, transformation, transposition

• using all of these we can stick in new genes to make a new outcome

• using these methods we can stick new genes into cells and then these cell then produce the protein

genetic engineering

hacking the genetic and gene expression of an organism

• one way to do this is by putting in recombinant dna (cutting a portion of 1 dna out and putting in a new dna from another species to create something)

recombiant dna

dna from more than 1 organism; has more than 1 species of dna from this

• can put this into an organism that makes it a transgenic organism bc it has genes from other organisms

genetic engineering insulin example

used to get insulin from pigs pancreases, so this is the other alternative to doing that so we dont have to kill lots of pigs

• creates a lot of transgenic organisms bc it has recombinant DNA

• due to the bacterium having the recombinant dna, the cell is now expressed as insulin

• allows us to create more human insulin effectively.

how can we cut out a specific part of a gene?

do this using restriction enzymes, "restriction endonucleases," or “molecular scaplels””

• they are a protein and they are endonucleases

• these endonucleases tend to cut at palindrom sequences

endonucleases

cut inside a dna sequence

how do estriction endonucleases work?

any place it finds a specific recognition site (this is a specific dna sequence), it will cut exactly there)

• has two ways it does this

  • stick end cutting

  • blunt end cutting

sticky end cutting

after these endonucleases cut the dna sequence they leave an overhang

• this allows us to control exactly how the piece will fit back in

  • g only to Cs

  • As only to Ts

blunt end cutting

when the endonucleases cut a sequence they dont leave an overhang creating 2 pieces of dna

palindromic sequence

same forwards and backwards

expression vector

its something that carries stuff that were trying to express

• it has a multicloning site (MCS)

• the part right before where u can put ur gene of interest, it has something that they took from the lac operon allowing u to be able to add lactose or something that looks like lactose

  • causes the plasmid to start synthesizing and transcription will take place

Multicloning site MCS

a short sequence containing multiple unique restriction enzyme recognition sites that r used for inserting dna into the plasmid

• allows for u to cut a certian gene with its specific polylinker

Molecular cloning

1. both foreign dna and a plasmid gene are cut with the same restriction enzyme. after they are cut, then they are left with these overhanging sticky ends

2. the sticky ends help us to ligate the foreign dna into the plasmid exactly into the right place

3. we often use dna ligases (a certian enzyme) to help when we ligate in this gene of interest into our plasmid

   1. dna ligases: tie together 2 pieces of dna (do oppo of restriction enzyme)

4. after they have been ligated, we have recombinant plasmids and we have to get those into cells somehow

   1. to get them into the cell we can do transformation

5. after we transform them we have to check which cells actually got transformed

   1. we can do this by doing a smear plate

   2. do this by seeing if the plasmid has amp resistance

once that plasmid goes through all this molecular cloning, it is then expressed as one of these bioengineering molecules because of the foreign dna that had been added

transfection

process by which you get the dna in a eukaryotic cell

• found ways to do this in the lab (these ways are like the loterry, only sometimes it will work)

  • electroporation

  • gene gun

  • microinjection

electroporation

basically use electric shock to shock the cell which causes pores to form and the gene enters

• sometimes dosent work well

gene gun

take gold beads coated in dna and shoot the beads using a gun into a plant cell and in a few cases get their dna inside the cell

microinjection

inject recombinant dna into a cell using a needle

elecrophoresis

seperating dna, protein, etc using a semi solid media based on charge and size

Polymerase Chain Reaction (PCR)

INVITRO - inside a thermal cycler

• cycle 1: starts with 1 strand of ds DNA

  • 1. denaturation: causes the strands to come apart

    • melting of the strands which is done by rasing the temp

  • 2. annealing: thermal cycler cools down causing this to happen

    • inside thermal cycler tube is the template dna, some primers, dna polymerase, and atcg’s

    • in this phase the primers bond on to our 2 ss DNA

  • 3. extension: dna polymerase does this

    • it starts adding on to the 3’ end. it synthesizes on 1 strand and on to the other strand going in the oppo direction and doing this gives us 2 copies of dna

  • after this cycle it does it again

• cycle 2: starts with 2 ds DNA

  • same thing occurs from cycle 1 and now we have 4x as much from where we started

  • continues going making lots of dna

• now with all of this dna u can do tests with them like electrophoresis, etc

primers

short segments of dna that help our dna polymerase to start copying

• complementary to ssDNA and tell the dna polymerase where to start

PCR equation

Nf = Ni x 2^n

• n = PCR cycles

TAQ polymerase

the dna polymerase that is used in PCR bc its able to withstand high heat

RT-PCR

reverse transcription - PCR; allows PCR of RNA

• take RNA then do reverse transcription and then do PCR

  • RNA does RT to make RNA and DNA, that DNA is used for PCR to make many DNA’s

DNA sequencing

determining the order of nucleotides AGCT in DNA

• older method: sanger dideoxy method

• newer method: modern chain extention method

sanger dideoxy method

worked by performing chain elongation with dye

• labled markers that stop dna replication

• dna fragments were then seperated using gel electrophoresis, with smaller fragments moving out first

• by reading colored dyes they could figure out the sequence

• was done by hand

modern chain extention method

works by taking a photo of the flourecent signals, washing them off, and continuing to extend the dna chain

• an automated machine performs this repetitive process allowing scientists to read the sequencing myst faster and more accurate

output and application of dna sequencing

• the end result is converting physical dna molecule into a graphical output that can be interperted (agct)

• this sequence info can then be used for various applications in genetic engineering such as

  • identifying henes, creating expression vectors, or developing treatments

gene therapy

• direct gene therapy using viral vectors

• xenotransplantation

• future potential: direct genetic engineering of humans

direct gene therapy using viral vectors

how it works: a func gene is packaged into a virus and delivered to patient cells through transfection. the viral vector carries the theraputic gene into the patients cells allowing them to express the missing or defective protein

• hasnt worked very well and viral delivery tends to cause inflamation and systemic side effects

xenotransplantation with genetically engineered pig organs

• pigs have organs around the same size as humans, but the human immune system naturally rejects them because they lack certian surface proteins that humans have

• many pigs carry viruses in their genome that cause problems, but scientists use genetic engineering to remove pig viruses and modify pig organ proteins making them moer like humans

  • this increases the transplant compatibility and rejection rate decreases

• prommising method

direct gene engineering of humans

doing genetic engineering directly on humans and not from another animal