In vivo gene cloning

Can you remember...

What we mean by in vitro and in vivo?

So far....

• You have found out about obtaining fragments of DNA by:

Using reverse transcriptaseUsing restriction endonucleases

• You can then make copies of these genes using which in vitro technique?

PCR

In vivo cloning can also be used to copy genes

Steps in in vivo cloningIn vitro to start with

Stage 1

Stage 2

Stage 3

Stage 4

Stage 1 – obtaining the DNA for the gene we want to clone, we looked at this last week

Stage 2 – is insertion of the DNA into a vector• A vector is required in order to transfer the

extracted gene into a chosen bacterial cell.

• An example of a vector is a plasmid i.e. a circular piece of DNA found in a bacterium.

How do we insert the gene into the plasmid? (refs p 21 booklet p249-251 A2 book)

• The plasmid is treated with the same restriction endonuclease enzyme as the extracted DNA.

• This means it has complementary sticky ends to the extracted DNA so they can be joined together by the enzyme DNA ligase.

On your plasmid

Colour in the sequence:

CGTAAT …this represents the gene for ampicillin resistance

AGAATTCT…this represents the gene for tetracycline resistance

Try this exercise with your pieces of human and plasmid DNA

(You will need to remember the recognition site for ECoR1. )

• Treat the human DNA with Eco R1 restriction endonuclease enzyme ( )

• Use your Eco R1 restriction enzyme i.e to cut the plasmid and produce sticky ends

complementary to your human gene. • Use your DNA ligase i.e. To join the sticky ends

of the plasmid with those of the human gene• You have now made a recombinant plasmid!!

• Adenoviruses and liposomes are other examples of vectors, apart from plasmids

LiposomeAdenovirus

Steps in in vivo cloningIn vitro to start with

Stage 1

Stage 2

Stage 3

Stage 4

Stage 3 – inserting the plasmid into bacterial cells

• Bacteria and plasmids are mixed in a medium containing calcium ions.

• This makes the bacterial cell wall more permeable to plasmids, which pass into the cytoplasm of the bacteria.

• Bacteria which contain the recombinant plasmid are said to be transformed

Steps in in vivo cloningIn vitro to start with

Stage 1

Stage 2

Stage 3

Stage 4

Firstly, we need to identify which bacteria contain the recombinant plasmids

How can we identify bacteria which have taken up the recombinant plasmids?

• We use marker genes on the plasmid. If these are expressed, we assume the bacteria are transformed. The marker genes code for:– Antibiotic resistance– Fluoresence– An enzyme

Antibiotic resistance markers (extra sheets)

• MARKER GENE eg antibiotic resistance

FOREIGNGENE eg insulin

+

Some bacteria transformed

Which of the two bacteria on the right would grow in the presence of an antibiotic?

Why?

How can the transformed bacteria be identified?

By culturing the bacteria on an agar plate which contains the antibiotic.

•

•

Transformed bacteria (contain the resistance gene)

NO GROWTH

Untransformed bacteria (no resistance)

GROWTH

Plasmids with 2 resistance genes can also be used (getting complicated and clever now!)

• Complete the first part of the exercise on using 2 marker genes

Ampicillin resistance gene

Tetracycline resistance gene

Bam H1 site

After the plasmid has been cut with Bam H1…..

Recombinant plasmid produced:

• Will the tetracycline resistance gene still function?

Ampicillin resistance gene

Human gene inserted into tetracycline resistance gene

But....

• Sometimes the original plasmid rejoins its sticky ends to itself without inserting the foreign gene

• complete these diagrams to show the three possibilities

a) contain the recombinantplasmid

b) contain the plasmid which has rejoined without the human gene

c) haven’t taken up any plasmids

So..we need to ensure we only select bacteria with the recombinant plasmid

• In this example – we first grow the bacteria on a plate containing ampillicin

• Which bacteria from a, b and c can grow on the ampicillin plate and why?

• a and b, as both groups of bacteria contain plasmids with the gene for ampicillin resistance. C will die as no plasmid.

• Copy the details on the plate below onto your ampicillin plate in the middle of page 24

Ampicillinplate

Then..• We take a replica of these colonies and grow

them on a tetracycline plate. • Which bacteria from a and b can grow on the

tetracycline plate and why?• b only as a are no longer resistant to

tetracycline as the target gene has been inserted into the tetracycline gene.

• Copy the details on the plate below onto your tetracycline plate

Tetracyclineplate

Only bacteria with the intact plasmid and both complete resistance genes can grow on this plate.

Ampicillinplate

Tetracyclineplate

Ampicillinplate

Tetracyclineplate

Original ampicillinplate

The bacteria which contain the recombinant plasmid are These – because they only grow on ampicillin and don’t grow on tetracycline, as the foreign gene has been inserted into their tetracycline resistance gene

Steps in in vivo cloningIn vitro to start with

Stage 1

Stage 2

Stage 3

Stage 4

Firstly, we need to identify which bacteria contain the recombinant plasmids

Finally...Stage 4 Growth / cloning (in vivo)

• The transformed bacteria, which have now been identified, are now grown on a large scale in a fermenter. They will secrete the gene product (e.g. insulin) into the medium they are growing in and it can then be extracted and purified.

Now test yourself...

• Complete the summary questions on page 253 of the text book

• Answer the exam question on page 8 of your booklet.

Genetic engineering question

a)(i) Codon; 1 (ii) Tyrosine; 1 (b) (i) Base sequence / codon (of DNA) is changed;Different (sequence of bases in) mRNA;Attracts different tRNA / anticodon;Different amino acid inserted into protein / polypeptide; 3 max

(ii) More than one base triplet / codon codes for one type of amino acid;

Suitable example / true for the third base of the codon; 2

(c) Endonuclease / restriction enzyme cuts DNA;Reference to specificity sticky ends / use the

same restriction enzyme on fragment and plasmid;

Ligase used to fix ends; 3

(d) Bacteria spread on agar to obtain separate colonies;Grown on agar containing ampicillin;Bacteria containing plasmid survive;For principles and detail of replica plating:Eg sterile filter paper / felt / nylon membrane;To obtain an exact copy;Placed on agar containing tetracycline;Bacteria growing on ampicillin, but not tetracycline containthe recombinant plasmids;Because foreign DNA has been inserted into the tetracycline gene; max 5 Total 15

Over the holidays….

1. Do the activity at the top of page 9 for yourself…Find out:• A couple of specific examples• A brief description of how the modification was carried out.• The specific benefits• Potential risksDo this for each of animals, plants and microorganisms2. Read about the ethical issues and the opposing views of the

humanitarian aspects and the anti-globalisation activists (p96/7 revision guide and application exercise p259-261)