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Why is the plasmid that contains the strongest promoter not always the best expression vector?

Question – 06

Describe a strategy for increasing the thermostability of a monomeric (single subunit) protein.

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Sixta Kovacek
Sixta KovacekLv2
28 Sep 2019

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Related questions

1. You have cloned a human gene along with its promoter and ribosome-binding site in a bacterial cloning vector and then introduced the recombinant plasmid into a bacterial host cell that supports the replication and segregation of the cloning vector. Unfortunately, even though you know the whole human gene sequence is present (by DNA hybridization); you cannot get expression of the gene. Which of the following is not a reason why there is no expression?

A. The vector cannot replicate

B. The human promoter is not recognized by the bacterial host cell

C. The ribosome-binding site is not recognized by the bacterial host cell

D. The human gene lacks introns, so it is not transcribed by the host bacterium

You need to express and purify a large amount of a protein for testing in an animal trial. The protein is larger than average, though not excessive so (~100 kDa, so the transcript is ~4,000 nucleotides long) and has an unusually acid isoelectric point (pI = 5). Propose and explain a strategy to clone, express, and purify this protein. [note: do not restate your answer for the previous question here, just state what factors you would consider when deciding on a cloning method. There is no single right answer for the choice of expression system or purification strategy; you should however be able to describe a rational approach and state why you chose that approach. Include some discussion of alternatives, such as “if A doesn’t work I will try B, and I think B is the next best choice because…”.

You only need to answer #4! The rest of the questions are there for relevancy.

1. To facilitate your study, you create a GFP tagged version of Tral. Which one of the following is not a step you would perform to accomplish this?

A. PCR the Tral gene from Drosophila genomic DNA.

B. Digest the PCR product and a GFP-containing plasmid.

C. Add the insert and vector together and add DNA ligase.

D. Transform the ligation mixture into Drosophila.

E. All of the above would be performed.

2. For this cloning, we need to include a _______________ promoter in the plasmid.

A. Bacteria

B. Drosophila

C. Neither

D. Both

3. You also speculate that various transcription factors could be regulating Tral expression. One such factor is Snail.

a. If Snail is a transcription factor activating Tral expression, which of the following could potentially be a function of Snail? Select the best answer.

A. Inhibit the binding of RNA polymerase at the Tral promoter.

B. Recruit a histone modifying protein that opens up the chromatin at the Tral gene.

C. Recruit DNA helicase at the origin of replication.

b. You want to determine whether Snail binds to the Tral promoter DNA. Which technique(s) would allow you to determine whether this occurs. Select all that apply.

A. Quantitative RT PCR

B. Chromatin Immunoprecipitation

C. Immunoprecipitation

D. Western blot

E. DNA footprinting

4. Now you want to determine the effect of the loss of Snail on Tral-GFP expression. IfSnail is an activator of transcription, draw/describe the expected results from a:

a. Northern blot

b. Q-RT PCR reaction

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