Bacterial Transformation Flashcards

You may prefer our related Brainscape-certified flashcards:
1
Q

What kind of protien structure is Insulin?

A

Quatenary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Tell me briefly about the insulin structure

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the two chains in insulin called?

A

Alpha chain and Beta chain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

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

A

2 seperate ones

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How does Insulin gene have no introns?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

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

A
  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.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is a fusion protien?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

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

A

Mehtionine, start codon

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

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…..

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

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

A

Transformed bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is a plasmid?

A

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

17
Q

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

A

Plasmids can be artifically made to deliver gene of interest.

18
Q

What do you need in making a recombinant plasmid

A
  1. DNA ligase
  2. Restriction ENzymes
  3. Gene of interest
  4. A plasmid vector
19
Q

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

A

Genetic code Universal

20
Q

Why does Plasmid have anitbiotic reistance gene

A

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

21
Q

What is a reporter GENE?

A

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

22
Q

Why iS DNA ligase used

A

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

Strong asf bonds frrfrrfrfr

23
Q

What are the steps for Bacterial transformation( Breif)

A

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

24
Q

What are the two ways bacteria can uptake a recombinant plasmid

A

VIA heat shock or electroporation

25
Q

What is heat shock?

A

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.

26
Q

What is electroporation?

A

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

27
Q

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

A

Prepare a platr with just nutrient Agar

28
Q

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

A

Introns

29
Q

Why do we ggotta even check about the reporter gene

A

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

30
Q

How is the bacteria grown

A

Binary Fission

31
Q

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

A

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

32
Q

How do we even get the Insulin A gene?

A

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.

33
Q

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

A

No

34
Q

What are the steps for bacterial transforation

A
  1. Do this cue card bro u forgot
35
Q
A