Gene fusions and CRISPR cas

Question 1

What is the difference between transcriptional and translational reporter fusions?

Answer 1

Transcriptional fusions use the promoter of the test gene, but the transcriptional machinery of the reporter gene.
Translational fusions use both the promoter and transcriptional machinery of your test gene, but are fused in the coding region of a reporter gene.

Question 2

How does crispr cas protect against bacteriophage attack?

Answer 2

Bacteria which have previously survived attack from bacteriophage can employ CRISPR-cas system to defend if an attack reoccurs.
CRISPR cas is immune system which encodes for cas nuclease, and contains spacer sequences which are chunks of bacteriophage DNA.
This allows for bacteriophage to be recognized, and initiates transcription of cas genes - providing RNA mediated nuclease attack on bacteriophage genome.

Question 3

Which three transcripts does the cas operon encode for?

Answer 3

Cas 9, tracrRNA and crRNA.

Question 4

Briefly describe how crispr-cas can be exploited as a sequence specific antimicrobial.

Answer 4

Genetically reprogram cas9 against desired genomic sequences - will cause lethal chromosomal lesions.
Can deliver Cas9 via a phagemid system.

Question 5

Summarise how all bacterial genetic assays can be used to identify the function of gene x (infective agent)

Answer 5

TMDH can be used to identify the Gene X is essential in condition B (infection)
Promoter Trap experiment to ensure gene is switched on during infection and colonization.
Lamda red can be used to create a null X mutant. Test to ensure null mutant means no infective phenotype.
Complement null mutant with wild type - Is complemented phenotype infective?