Bacterial Transformation

Question 1

What kind of protien structure is Insulin?

Answer 1

Quatenary

Question 2

Tell me briefly about the insulin structure

Answer 2

It is a small protien with two seperate amino acid chains called Alpha chain and Beta chain

Question 3

What are the two chains in insulin called?

Answer 3

Alpha chain and Beta chain

Question 4

How many recombinant plasmids do you need to make for Insulin production

Answer 4

2 seperate ones

Question 5

How does Insulin gene have no introns?

Answer 5

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.

Question 6

Why do you need Beta galactodsidase in Insulin Production and one other reason.

Answer 6

1. 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.
2. We have it because the promoter region is found in the Lacz gene, so it starts transcription.

Question 7

What do you do now to identify the transformed bacteria with the plasmid including the LacZ gene? And why does that happen

Answer 7

You use a agar plate that contains the organic compound called X-gal, this turns the bacteria blue in colour when reacted.

Question 8

What is a fusion protien?

Answer 8

A single protien that is expressed when two genes are together.

Question 9

Can scientists turn on and off the the whole fusion gene when they want to?

Answer 9

Yes, because a repressor protien is often binded, and can be activated to detatch by adding Allolatose, which turns the Gene on.

Question 10

What is added to the start of the Insulin A gene?

Answer 10

Mehtionine, start codon

Question 11

How do they seperate the two a chain from b-galactodase.

Answer 11

Because methionine was added and doesnt affect the protiens structure of function. You use a chemical to break down.

Question 12

Why tf does RNA polymerase jus run across the fusion gene without stopping?

Answer 12

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.

Question 13

Both THe plasmids and gene that is being cloned must both be…

Answer 13

Cut using the same restriction enzymes, so the complementary sticky ends connect.

Question 14

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…..

Answer 14

is used to distinguish the transformed bacteria that includes a recombinant plasmid

Question 15

What do you call bateria that has taken up foreign DNA via a plasmid

Answer 15

Transformed bacteria

Question 16

What is a plasmid?

Answer 16

A small circular DNA strand
found in Bacterial cells that indroduces foreign DNA into an organism as a VECTOR.

Question 17

How tf bro did the Plasmid already have antibiotic gene in it??

Answer 17

Plasmids can be artifically made to deliver gene of interest.

Question 18

What do you need in making a recombinant plasmid

Answer 18

1. DNA ligase
2. Restriction ENzymes
3. Gene of interest
4. A plasmid vector

Question 19

Why can we use human DNA and be able to put it into a plasmid?

Answer 19

Genetic code Universal

Question 20

Why does Plasmid have anitbiotic reistance gene

Answer 20

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

Question 21

What is a reporter GENE?

Answer 21

Creates a easily identifiable phenotype to be observed which is often not express when the gene of interest has sucessfully been combined.

Question 22

Why iS DNA ligase used

Answer 22

It is used to form phosophodiester bonds in the sugar phosphate backbone with the gnee of interest and plasmid.

Strong asf bonds frrfrrfrfr

Question 23

What are the steps for Bacterial transformation( Breif)

Answer 23

1.Uptake of recombinant plasmid
2.Antibiotic selection
3.Bacterial identfication
4.Protien produced

Question 24

What are the two ways bacteria can uptake a recombinant plasmid

Answer 24

VIA heat shock or electroporation

Question 25

What is heat shock?

Answer 25

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.

Question 26

What is electroporation?

Answer 26

Pass through a electrical current through solution, which makes the memebrane permiable

Question 27

What could be the negative control for an expermient like this?

Answer 27

Prepare a platr with just nutrient Agar

Question 28

What about the gene of interest do you gotta make sure it cant have

Answer 28

Introns

Question 29

Why do we ggotta even check about the reporter gene

Answer 29

Because plasmids dont always from an recombinant plasmid, they dont take up the gene of interest everytime, creating undesired plasmids.

Question 30

How is the bacteria grown

Answer 30

Binary Fission

Question 31

What do you do now after finding colony that dont express reporter gene?

Answer 31

After identifying the colonies with transformed bacteria with recombinant plasmid. It can then be extracted and purified.

Question 32

How do we even get the Insulin A gene?

Answer 32

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.

Question 33

do we use two different restriction enzymes when cutting the Insulin A/B gene?

Answer 33

No

Question 34

What are the steps for bacterial transforation

Answer 34

1. Do this cue card bro u forgot