Week 10 Tuesday

Question 1

What are the reasons genome editing can fail and you get zero colonies?

Answer 1

a bad transformation, or you have CRISPR killing but no homologous recombination

Question 2

What are the reasons genome editing can fail and you get colonies that are without the edit?

Answer 2

then they might not have gotten the edit because CRISPR cleaves, but the cells stitch back together (they escaped CRISPR)

Question 3

How do we test if the genome editing succeeded but the colonies are not orange?

Answer 3

can sequence the genome or do PCR (amplify the chunk we care about) to see if it was a success

Question 4

Why would a PCR come back positive for the correct pathway but the colonies not be orange?

Answer 4

that could be from something in the pathway going wrong. Maybe the genes aren’t being expressed on the plasmid or maybe the final enzymes are not active in the cell.

Question 5

How do we know how much beta carotene our cells are making?

Answer 5

Qualitative methods – visual, color, morphology, microscopy, etc…
Quantitative methods – spectroscopy (light, fluorescence, mass), chromatography (separation principles)

Question 6

Why do we use a wavelength of 450?

Answer 6

Because it is the wavelength of orange.

Question 7

Why might UV vis not work?

Answer 7

media is a similar color to the beta carotene

Question 8

What are the three enzymes required for the beta-carotene pathway?

Answer 8

synthase, desaturase, cyclase

Question 9

Why might discovering side products be good?

Answer 9

Discovering side products in pathway could remove bottlenecks or lead to new innovation