Gene captain and gene spike work together to promote gene expression. I want to
ID: 54733 • Letter: G
Question
Gene captain and gene spike work together to promote gene expression. I want to express these two gene products in cells and run a GFP reporter assay to determine if they work independently, additively, or synergistically to enhance the expression of gene awesome.
A) How will you generate an expression vector for each of these genes? Write this out in detail. Start with the DNA that you will be using, state what vector you will clone into and explain your strategy for getting the DNA into the vector including any enzymes you may use (Just explain in detail for one gene and I will assume you can express the other vector as well). (DO NOT USE THE PREVIOUS CHEGG RESPONSE FOR THIS ONE. IT'S BEING ASKED A SECOND TIME FOR A REASON).
Explanation / Answer
We can do this by generating three types of recombinant DNA vectors:
1. In one expression vector, under the control of a constitutive promoter, the gene (DNA) encoding Gene Captain is cloned.
Also at another site, gene encoding GFP is inserted under the control of a promoter inducible by Gene Captain & gene spike -gene products. (with regards to the choice of vector, choose an expression vector that helps in negative selection; you can also choose vectors that enable blue-white screening) .
2. In second type, under the control of a constitutive promoter, the gene (DNA) encoding Gene Spike is cloned.Also at another site, gene encoding GFP is inserted under the control of a promoter inducible by Gene Captain & gene spike -gene products.
3. In third type, under the control of constitutive promoters, both the genes of Gene Captain & Gene Spike are cloned. Also at another site, gene encoding GFP is inserted under the control of an inducible promoter.
Now, for observations:
GFP expression in type I
+ve
Gene captain can function individually
GFP expression in type I
-ve
Gene captain cannot function individually
GFP expression in type II
+ve
Gene spike can function individually
GFP expression in type II
-ve
Gene spike cannot function individually
GFP expression in type III
+ve ( -ve for type I & II)
Gene captain & gene spike function together
GFP expression in type III
+++
Gene captain & gene spike function together in a synergistically
For the choice of plasmid vectors, let us choose pCMV-AC-GFP, where the GFP gene is present under the control of constitutive promoter pCMV; a lot of Restriction sites are engineered between the promoter & the GFP gene. Our DNA (consisting of the gene for Gene Captain & followed by the inducible promoter- this is for 1st type of constructs) is inserted in one of those sites; let us use Hind III & XhoI ( buffer compatibility in NE buffer). Use of two restriction enzymes enable directional cloning.
GFP expression in type I
+ve
Gene captain can function individually
GFP expression in type I
-ve
Gene captain cannot function individually
GFP expression in type II
+ve
Gene spike can function individually
GFP expression in type II
-ve
Gene spike cannot function individually
GFP expression in type III
+ve ( -ve for type I & II)
Gene captain & gene spike function together
GFP expression in type III
+++
Gene captain & gene spike function together in a synergistically
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