Bacterial Transformation Flashcards
What kind of protien structure is Insulin?
Quatenary
Tell me briefly about the insulin structure
It is a small protien with two seperate amino acid chains called Alpha chain and Beta chain
What are the two chains in insulin called?
Alpha chain and Beta chain
How many recombinant plasmids do you need to make for Insulin production
2 seperate ones
How does Insulin gene have no introns?
They used the genetic code, degenerate. By synthesising a gene that has the any of the correct triple codons that code for the specific amino chain sequence for the subunit chain.
Why do you need Beta galactodsidase in Insulin Production and one other reason.
- Because in a cell, enzymes often degrade any small unfolded amino acid peptides in bacteria. herefore when adding that gene, it protects it from being broken down.
- We have it because the promoter region is found in the Lacz gene, so it starts transcription.
What do you do now to identify the transformed bacteria with the plasmid including the LacZ gene? And why does that happen
You use a agar plate that contains the organic compound called X-gal, this turns the bacteria blue in colour when reacted.
What is a fusion protien?
A single protien that is expressed when two genes are together.
Can scientists turn on and off the the whole fusion gene when they want to?
Yes, because a repressor protien is often binded, and can be activated to detatch by adding Allolatose, which turns the Gene on.
What is added to the start of the Insulin A gene?
Mehtionine, start codon
How do they seperate the two a chain from b-galactodase.
Because methionine was added and doesnt affect the protiens structure of function. You use a chemical to break down.
Why tf does RNA polymerase jus run across the fusion gene without stopping?
This is because when ECOR1 was used to cut out the LacZ gene, the stop codon and part of the operon was removed, so when it binded with Insulin A gene, there is no stop codon to stop transcription.
Both THe plasmids and gene that is being cloned must both be…
Cut using the same restriction enzymes, so the complementary sticky ends connect.
In gene cloning, in many cases a plasmid is selected that has two genes for antibiotic resistance. In such cases, the second gene for antibiotic resistance…..
is used to distinguish the transformed bacteria that includes a recombinant plasmid
What do you call bateria that has taken up foreign DNA via a plasmid
Transformed bacteria
What is a plasmid?
A small circular DNA strand
found in Bacterial cells that indroduces foreign DNA into an organism as a VECTOR.
How tf bro did the Plasmid already have antibiotic gene in it??
Plasmids can be artifically made to deliver gene of interest.
What do you need in making a recombinant plasmid
- DNA ligase
- Restriction ENzymes
- Gene of interest
- A plasmid vector
Why can we use human DNA and be able to put it into a plasmid?
Genetic code Universal
Why does Plasmid have anitbiotic reistance gene
When identifying the bacterial colonies, using a agar plate consisting the antibiotic can help deferentiate the bacteria that has sucessfully been transformed containing a plasmid. NOTE you cant confirm if its recombinant
What is a reporter GENE?
Creates a easily identifiable phenotype to be observed which is often not express when the gene of interest has sucessfully been combined.
Why iS DNA ligase used
It is used to form phosophodiester bonds in the sugar phosphate backbone with the gnee of interest and plasmid.
Strong asf bonds frrfrrfrfr
What are the steps for Bacterial transformation( Breif)
1.Uptake of recombinant plasmid
2.Antibiotic selection
3.Bacterial identfication
4.Protien produced
What are the two ways bacteria can uptake a recombinant plasmid
VIA heat shock or electroporation
What is heat shock?
It is when you place the bacteria and the plasmid in a calcium ion solution and heat it up quickly.
This makes the cell membrane in the bacteria permiable for the plasmid to enter.
What is electroporation?
Pass through a electrical current through solution, which makes the memebrane permiable
What could be the negative control for an expermient like this?
Prepare a platr with just nutrient Agar
What about the gene of interest do you gotta make sure it cant have
Introns
Why do we ggotta even check about the reporter gene
Because plasmids dont always from an recombinant plasmid, they dont take up the gene of interest everytime, creating undesired plasmids.
How is the bacteria grown
Binary Fission
What do you do now after finding colony that dont express reporter gene?
After identifying the colonies with transformed bacteria with recombinant plasmid. It can then be extracted and purified.
How do we even get the Insulin A gene?
1.We get a mRNA strand that has been transcribed by a insulin gene from a cell
2.Then we use a enzyme called reverse transcriptase which makes a DNA strand called cDNA
3.and then Voilla you cut the gene of interest out with no introns at all.
do we use two different restriction enzymes when cutting the Insulin A/B gene?
No
What are the steps for bacterial transforation
- Do this cue card bro u forgot
