Lecture 28

Question 1

Screening and reporting gene

Answer 1

Reporter genes produces a measurable product it indicates successful recombination (quantitative or qualitative). The MCS is within reporter gene

Question 2

Lac Z

Answer 2

If reported gene functions correctly it produces B-galactosidase. When X-gal is added to agar plate, it will produce a blue compound and bacterial colony turns blue. In recombinant, RG has been cut and gene has been inserted therefore, no blue colony. X-gal on it’s own is clear.

Question 3

Blue-white screen drawbacks

Answer 3

Good for viewing many colonies at once. But, there are undesirable remnants of lac Z gene where there won’t be enough B-galactose for turning it blue therefore more subcloning needed.

Question 4

Colony PCR

Answer 4

Have colony plate (don’t know whether it’s been transformed or not), you split it in half where ½ goes into a labelled tube and the other goes onto labelled plate. Perform PCR on the tubes and electrophoresis. Non- recs. are smaller therefore, travel further and then you can pick colonies for sequencing.

Question 5

Colony PCR

Answer 5

Relatively fast and cheap, many clones can be screened at a time. Might not work and needs sequence confirmation.

Question 6

Restriction digestion

Answer 6

Simple to set up, requires grow up of bacterial cells and plasmid extraction, needs sequence confirmation. Colony divides into tube and plate. Plasmids extracted, restriction digest occurs where RE cut before promoter and after MCS. Recombinant = larger PCR = shorter distance between bands. Pick colonies for sequencing.

Question 7

Sequencing

Answer 7

Direct sequence confirmation, requires grow up of bacterial cells and plasmid extraction. Slow and expenses add up. Longer and more expensive to sequence colony all at once. When nucleotide added to complementary strand, a fluorescent signal is sent and they are detected. Allows exact sequence to be mapped and you can match it to what you’d expect.

Question 8

CRISPR/cas 9

Answer 8

CRISPR is apart of a bacteria’s defence system. Guide RNA lead Cas 9 to a specific DNA sequence and it contains a complementary sequence to target DNA. Cas 9 creates a double strand break at target site which the cell tries to repair through non-homologous end joining (may introduce mutations) or homologous-directed repair (precise insertion of correction using repair template). Can create synthetic GRNA to cut out target gene so that it can be replaced by functional gene.

Question 9

Sickle cell and CRISPR

Answer 9

Caused by point mutation in the B globin gene. Leads to sickle shapes RBC. Previous treatment aimed to manage symptoms incl. anaemia, pain and infections. Hematopoietic SC can be edited using CRISPR/Cas 9 to reactivate foetal Hb production to compensate for defective adult Hb. Can correct B globin mutation or remove BCLIIA which represses foetal Hb production. Edited cells reinfused back into patient.