Genetic engineering (recombinant DNA technology)

Steps1. Obtain gene2. Put the gene into the vector3. The vector carries the gene into the

recipient cell4. The recipient cell expresses the gene.

Restriction enzymes.

Cut the backbone of the DNA moleculeMany different typesEach one cuts at a specific sequence, the restriction siteSome stagger where they cut the backbone to give DNA with “sticky ends”

A T C C G TT A G G C A

A T C C G T T A G G C A

A C G A T C C G T A T C T G C T A G G C A T A G

A C G A T C C G T A T C

T G C T A G G C A T A G

Recombinant DNA = DNA from different organisms that has been joined together

Plasmids•Small circular pieces of DNA that are separate from the main chromosome.•Bacteria can exchange plasmids by a process called conjugation

Genetic engineering (recombinant DNA technology)

Steps1. Obtain gene2. Put the gene into the vector3. The vector carries the gene into the

recipient cell4. The recipient cell expresses the gene.

Obtain gene

1. Locate it in the genome using a probe then cut it out using restriction enzymes

2. Synthesis Gene using an automated polynucleotide sequencer

3. Use the mRNA from a cell that expresses that gene as a template to make a DNA copy (cDNA) of the gene (to do this you need an enzyme called reverse transcriptase)

NB – gel electrophoresis is often then used to check the DNA is the same size as the piece you think it is….

Place gene in vector

1. Usually put into a cut plasmid and sealed with ligase

2. Plasmid will often contain regulatory sequences to make sure the gene is expressed

3. Viruses can also be used as vectors.

Put the vector into the recipient cell

1. Electroporation

2. Microinjection

3. Viral transfer

4. Use Agrobacterium tumefaciens and the Ti plasmid (plants)

5. Liposomes

6. (heat shock)

How could you check that the recipient has taken up the plasmid?

How could you check that the recipient has taken up a the plasmid into which the new gene has been successfully inserted?

How could you check that the recipient has taken up the plasmid?

•Bacteria that contain a successfully transformed plasmid will be resistant to ampicillin but not tetracycline.

•These bacteria can be selected using replica plating

No antibiotics

Plate with ampicillin Plate with tetracycline

Transformed bacteria can now be identified using replica plating

Recombinant DNA Transgenic bacteria

Precursor molecules - Produced in the rice grain

Phytoene

Lycopene

Beta Carotene

Phyotene synthetase (gene from daffodil)

Crt 1 enzyme (gene from a bacterium)

Enzymes present in the rice grain