In Vivo Cloning

Question 1

What does In Vivo mean?

Answer 1

Transferring fragments to a host cell using a vector

Question 2

What does DNA ligase do?

Answer 2

Links sticky ends together by their sugar phosphate backbone

Question 3

What binds to DNA and where does it bind to during preparation of the fragment for insertion?

Answer 3

RNA polymerase attaches to DNA near the gene at the promoter region

Question 4

Describe how the process of transcription is started.

Answer 4

Nucleotide bases of the promoter attach to RNA polymerase and transcription factors, beginning transcription.

Question 5

Name the region at which RNA polymerase is released from the DNA as transcription is completed.

Answer 5

Terminator

Question 6

What is a vector?

Answer 6

A small piece of DNA that a foreign DNA fragment can be inserted into.

Question 7

Give an example of a vector.

Answer 7

Plasmid

Question 8

Name the process where a plasmid is put into a host cell.

Answer 8

Transformation

Question 9

Name 2 things that are done to increase membrane permeability when putting plasmids back into a bacterial cell.

Answer 9

Increased temperature
Higher concentration of calcium ions

Question 10

Give a characteristic coded for by a gene found in most plasmids

Answer 10

Antibiotic resistance

Question 11

Name 3 things that are mixed together during insertion.

Answer 11

Bacterial cells
Calcium
Plasmids

Question 12

Give 3 reasons why not all bacterial cells will possess DNA fragments with the desired gene after the plasmid has been inserted.

Answer 12

Only a few bacterial cells will take up plasmids when mixed together
Some plasmids will close up without incorporating the DNA fragment
Sometimes the DNA fragment ends join together to form its own plasmid

Question 13

Describe how we can identify bacterial cells that have taken up the inserted plasmid.

Answer 13

Grow the bacteria on a medium containing an antibiotic.
The antibiotic resistance gene is found in plasmids only and therefore the bacteria that survive must contain the plasmid

Question 14

What is a gene marker used for?

Answer 14

To identify which plasmids have taken up the DNA fragment.

Question 15

Give 3 things that could be gene markers.

Answer 15

Antibiotic resistance
A fluorescent protein
An enzyme

Question 16

What happens to gene markers if the DNA fragment is present?

Answer 16

It is disrupted.

Question 17

If the DNA fragment has been inserted into a gene successfully, describe what we would see if a fluorescent marker had been used.

Answer 17

The bacterial cell will not glow so therefore can be identified.

Question 18

If the DNA fragment had not been inserted into a gene successfully, describe what we would see if a fluorescent marker had been used.

Answer 18

The bacterial cell will glow

Question 19

What is replica plating?

Answer 19

A technique used where bacteria are grown on 2 different petri dishes.
One petri dish contains ampicillin, an antibiotic, and bacteria that doesn’t grow on this plate contains a plasmid with a DNA fragment.
Bacteria that do grow on the other plate do contain the plasmid.

Question 20

What do we do once bacteria containing the plasmid have been identified?

Answer 20

They are cloned. This means that the plasmid is also cloned.