21.2 In vivo cloning

Question 1

What does in vivo mean

What about in vitro

In terms of DNA fragments

Answer 1

Transfer the DNA fragments into a host cell using a vector

In vitro, use polymerase chain reaction

Question 2

Restriction endonucleases leave sticky ends, how

Answer 2

At recognition site the restriction endonuclease cuts out a sequence of DNA nucleotides called a fragment.

These DNA nucleotide bases are complimentary to DNA from another source cut out with the same restriction endonuclease.

This means the single stranded end of any fragment can be joined to another if the complimentary bases line up.

These can bind together to make recombinant DNA, as DNA ligase joins them.
DNA ligase joins the phosphate-sugar framework of both sections of DNA

Question 3

Why are sticky ends left by restriction endonucleases important

Answer 3

They allow us to combine DNA of two organisms.

These sticky ends must then be modified to ensure transcription of these genes can occur

Question 4

What are the stages of in vivo cloning

Answer 4

• DNA fragments are made for gene of interest
• Insert DNA fragment into a vector
• Transform a host cell with the vector
• Identify transformed cells
• Grow the host cell

Question 5

To prepare DNA for insertion, the sticky ends must be modified so transcription can occur.

This is done by adding a promoter region: Explain

Also done by adding terminator region

Answer 5

• Add promoter region at the start of DNA fragment.
• This is a sequence of DNA nucleotide bases added so that RNA polymerase can bind to it and enable transcription to occur
• Terminator region is added at end of gene
  Causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA

Question 6

What is a vector

What are the most common ones and why

Answer 6

Something to carry isolated DNA into host cell

Plasmids in bacterial cell are separate loops of DNA, and almost always contain genes for antibiotic resistance

Question 7

What is the process of inserting DNA into vector

Answer 7

• Use the same restriction endonuclease as before to cut open plasmid.
• We do this because it cuts at the same sequence of bases
• This creates the same sticky ends so it means the DNA fragment sticky ends you have are complimentary to those so they bind
• Use enzyme ligase to stick the DNA in place by catalysing formation of phosphodiester bonds between phosphate and sugar

Question 8

What is transformation

Answer 8

The process of moving DNA from vector into host cell

Question 9

What do we do to the host cell to allow vector to be inserted in

Answer 9

The cell membrane needs to become more permeable

So mix host cell with Ca2+ ions and heat shock it

This enables vectors to enter the host cells cytoplasm

Question 10

Why don’t all bacterial cells contain the desired DNA fragments when transforming them into host cell cytoplasm

Answer 10

• Only a few bacterial cells actually take up the plasmids when they’re mixed together and heat shocked
• Some plasmids close again without incorporating the DNA fragment
• Sometimes the DNA fragment ends join together to make its own plasmid

Question 11

Where do bacteria have mechanisms for antibiotic resistance

What is R plasmid

Answer 11

• Bacteria have mechanisms for antibiotic resistance in plasmids, eg producing an enzyme that breaks down antibiotic before it damages the bacteria.
• R plasmid contains genes for resistance for two antibiotics, ampicillin and tetracycline

Question 12

How do we find out which bacteria have taken up the plasmids using this information

Process

Answer 12

• Use gene for antibiotic resistance which should be unaffected by the introduction of the new gene
• Grow all bacteria on medium containing antibiotic ampicillin
• The bacterial cells that have taken up plasmids will have antibiotic resistance to this
• So these ones will break it down and survive whilst others will die

So this method shows what bacteria have taken up the plasmids

Question 13

Although we now know what bacteria have taken up the plasmids, not all of the plasmids actually contain the gene we want.

How do we find out which ones did take up the gene

Answer 13

Marker genes

They all involve using a second separate gene on plasmid which is easily recognisable from others

Question 14

How would a marker gene be recognisable

Answer 14

• Antibiotic resistance
• It could make a fluorescent protein which can easily be seen
• It may produce an enzyme whos action can be identified

Question 15

How would you use antibiotic resistant genes as markers

Called replica plating

Answer 15

If the plasmid has two genes for resistance to ampicillin and tetracycline, cut open plasmid at the tetracycline part.

• Meanwhile get DNA from cell the manufactures desired protein
• Use restriction endonuclease to cut plasmid and DNA leaving sticky ends of DNA fragment

The plasmid previously can incorporate the sticky ended DNA fragment into it where the tetracycline resistant gene was previously.

This means the bacteria with the DNA is no longer resistant to tetracycline.

So grow on culture with tetracycline and they will die, so we can spot by comparing the plate with the previous one used to see which bacteria is missing and this would be them.

Question 16

How are fluorescent markers used to identify which plasmids contain DNA fragments

Answer 16

• Transfer gene from jellyfish into plasmid which produces a green fluorescent protein called GFP
• The gene to be cloned can be transplanted into the GFP gene
• So bacterial cells that haven’t taken up the DNA fragment will continue to glow as that gene isn’t damaged.
  Bacterial cells that have taken up the DNA fragment will not be able to produce the fluorescent protein

Question 17

How are enzyme markers used to identify which plasmids took up the DNA fragments

Answer 17

• Gene that produces the enzyme lactase
• Lactase can turn a colourless substrate blue
• So transplant the required gene into the gene that makes lactase
• So colonies from gene containing DNA fragments will not produce lactase so won’t turn blue