Microbial toolbox

Question 1

Agrobacterium

Answer 1

Agrobacterium takes advantage of cuts in plant
Inserts tumour inducing plasmid into plant cell, integrated into plant cell DNA
Rapid multiplication of infected cell = crown gall disease

Harmful tumour inducing gene is replaced with gene of interest in genetic modification, bacteria doesn’t know and delivers plasmid as usual

Question 2

Cre Lox system

Answer 2

Two components:
- Cre (cyclization recombination) recombinase (cut and paste DNA)
- Recognition site loxP (locus of crossing over bacteriophage P1)

adapted from P1 bacteriophages
Translocation: separate pieces
Inversion: opposite direction
Deletion: same way

Question 3

Virus mediated gene transfer

Answer 3

Gene of interest is inserted into the nucleic acid of the virus (adenovirus, retrovirus or lentivirus)
Virus enters cell via fusion
Releases core
Reverse transcription (into DNA) > integrated into genome

Question 4

Molecular cloning

Answer 4

Restriction enzymes:
- Haelll = blunt end
- BamHI = sticky end
Ligase
- T4 DNA ligase

Question 5

Humulin

Answer 5

part A and part B are inserted into an expression vector that is inserted into two cells. Bacteria are grown in a bio-reactor. The gene inserted contains beta-galactosidase for screening. Once bacteria with the expression vector are isolated, the product is purified (beta-galactosidase is removed) and the two parts are joined with a disulphide bond.

Question 6

Screening

Answer 6

Blue-white colony screening: inserting gene into beta-galactosidase gene and see if they turn white
PCR: primer is specialized for GOI, if it amplifies then it is successful