Question

In an experiment, E.coli cells have been genetically modified through site directed mutagenesis. This is a process through which specific locations in an organism’s genome can be altered in order to study the function of such locations, or proteins coded for by genes found in a location. There are many techniques available for site directed mutagenesis. (You have access to PCR ingredients, any primer you wish, laboratory equipment, Lambda phage, and the ability to alter Lambda genome, a commercially available plasmid, such as Puc19, E.coli K-12, plates, media, etc..)

5. You are interested in studying a bacterial promoter to see if heat-shock stress will activate this promoter. Design a gene such that you can test your hypothesis (your design must include all the necessary portions of a gene!) (3 points)

Answer 1

Aim: To check whether the given promoter is sensitive to heat shock
Requirements: PCR components, cloning kit, competent cells, etc...
Procedure:
i. PCR out the desired promoter using specific primers
ii. Coone the promoter into a cloning vector.
iii. Clone a reporter gene (GUS or GFP) downstream to the promoter
iv. Transform the construct into competent cells
v. Screen for positive clones and confirm them
vi. Give heat shock to the positive colonies and look for their phenotype

Inference:
If the promoter is sensitive to heat shock, we would observe the expression of the reporter gene (we can observe GUS expression by GUS -assay. We can observe GFP expression by fluorescence microscopy)
If the promoter is not sensitive to heat shock, we would not observe the expression of the reporter gene.