In Vivo Gene Cloning - The Use Of Vectors

Question 1

What are the two ways in which a gene can be cloned for medial or commercial use

Answer 1

In vivo - transferring the fragments to a host cell using a vector

In vitro - using the polymerase chain reaction

Question 2

How do we use sticky ends to

Combine DNA from different sources

Answer 2

Restriction endonuclease cuts out a dna fragment

DNA from other source is added ( DNA from other source cut with same restriction endonuclease)

DNA Ligase joins the two sections

Making recombinant DNA

Question 3

Why are sticky ends important

Answer 3

Because provided the same restriction is used, we can combine the DNA of one organism with that of any other organism

Question 4

What are the sequences of DNA that are cut by restriction endonucleases are called

Answer 4

Recognition sites

Question 5

How is the DNA fragment prepared for insertion

Answer 5

It involves the addition of extra lengths of dna.

Question 6

What happens for the the transcription of any gene to take place

Answer 6

the enzyme that synthesise mRNA (rna polymerase) must attach to the DNA near a gene

Question 7

What’s the binding site for rna polymerase

Answer 7

Region of DNA, known as promoter

Question 8

What is essential if we want our dna fragment to transcribe mRNA in order to make a protein

Answer 8

That we attach it to the necessary promoter region to start the process

Question 9

What’s a terminator

Answer 9

releases rna polymerase and ends transcription.

Question 10

Why do we need a terminator

Answer 10

To stop transcription at the appropriate point

Question 11

What is the vector used for. Give an example

Answer 11

Transport the DNA into the host cell

A plasmid

Question 12

Where are the endonucleases used to do in the plasmid

Answer 12

They are used at one of the antibiotic resistance genes to break the plasmid loop

Question 13

The restriction endonuclease used for the plasmid is the same as the one that cut of the DNA fragment. What does this ensure

Answer 13

That the sticky ends of the opened up plasmid are complimentary to the sticky ends of the DNA fragment.

Question 14

What happens when the DNA fragments are mixed with with opened up plasmids

Answer 14

They become incorporated into them

Where they are incorporated, the join is made permanent using the enzyme DNA ligase. These plasmids now have recombinant dna

Question 15

What happens during transformation

Answer 15

Introduction of DNA fragment into suitable host cell

Question 16

Describe transformation

Answer 16

It involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.

The calcium ions, and changes in temperature, make the bacterial membrane permeable, allowing plasmids to pass through the cell surface membrane into the cytoplasm

Question 17

What are the reasons for not all bacterial cells possessing the DNA fragments with the desired gene for the protein.

Answer 17

Only a few bacterial cells take up the plasmids when the two are mixed together

Some plasmids will have closed up again without incorporating the DNA fragment

Sometimes the DNA fragment ends join together to form its own plasmid

Question 18

The first task is to identify which bacterial cells have taken up the plasmid. How do we do this

Answer 18

Using the fact bacteria have evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium. The genes for the production of these enzymes are found in plasmids

Question 19

Describe the process of finding out which bacterial cells have taken up the plasmids entail using the gene for antibiotic resistance, which is unaffected by the introduction of the new gene

Answer 19

All the bacterial cells are grown on a medium that contains the antibiotic ampicillin

Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance

These bacterial cells are able to break down the ampicillin and therefore survive

The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die

Question 20

What do marker genes do

Answer 20

They identify whether a gene has been take up by bacterial cells. They all involve using a second, separate gene on the plasmid.

Question 21

How is the second gene easily identifiable for one reason or another. For example what…?

Answer 21

It may be resistant to an antibiotic

It may make a fluorescent protein that is easily seen

It may produce an enzyme whose action can be identified

Question 22

What does replica plating do

Answer 22

It identifies those cells with plasmids that have taken up the new gene

It uses the other antibiotic resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene.

Question 23

What’s a positive of replica plating

Answer 23

It is possible to identify living colonies of bacteria containing the required gene

Question 24

How are fluorescent markers used from the transfer of a gene from a jellyfish into the plasmid

Answer 24

The gene in question produces a green fluorescent protein. (GFP

The gene to be cloned is transplanted untinthr centre of the GFP gene.

Any bacterial cell that has been take up the plasmid with the gene that is to be cloned will not be able to produce GFP

Bacterial cells that have not taken up the gene will continue to produce GFP and to fluoresce. Unlike the cells that have not taken up the gene, these cells have taken it up will not fluoresce

As the bacterial cells with the desired gene are not killed, there is no need for replica plating.

Results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce. This makes the process more rapid

Question 25

Describe how the enzyme marker lactase works

Answer 25

It will turn a Particular colourless substrate blue.

Again, the required gene is transplanted into the gene that makes lactase.

If a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase.

Therefore when these bacterial cells are grown on the colourless substrate they will be unable to change its colour.

Where the genes has not been take up by the bacteria, they will not turn the substrate volume. These bacteria can be discounted

Question 26

T

Answer 26

To show which cells (bacteria) have taken up the plasmid (gene)

Question 27

X

Answer 27

Results can be obtained more easily and quickly - because, with antibiotic resistance markers, the bacterial cells with the required gene are killed, so replica plating is necessary to obtain the cells with the gene. With fluorescent gene markers, the bacterial cells are not killed and so there is no need to carry out replica plating