lpullela2424
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How to increase gene expression of HSP in E.Coli bacteria

Postby lpullela2424 » Tue Jul 24, 2018 11:47 am

Hi! I would like to conduct an experiment part of which involves increasing the gene expression of heat shock proteins (specifically DnaK, GroEl, and GrpE) in E. Coli bacteria. What is the best way to manipulate the gene expression of EACH of these proteins independently? Is there an organism more feasible to work with than E. Coli for this task? Thank you so much!

SciB
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Re: How to increase gene expression of HSP in E.Coli bacteria

Postby SciB » Thu Aug 02, 2018 1:58 pm

Hi,

E coli is the workhorse for bacteria-based research especially since the K12 lab strain used in most experiments does not cause human disease.

I am not familiar with bacterial heat-shock proteins, but I think one at least is similar to the mammalian Hsp90. From my coursed in microbiology I remember that bacteria use sigma factors to regulate gene transcription, but I don't know what regulates the sigma factors.

If you are considering a project of this difficulty, I would assume that you have access to a micro lab and the necessary skills to do these experiments--correct? I know a little about the three gene products you mentioned and they have all been studied pretty thoroughly so the gene expression regulation info must be out there somewhere. Here's a paper I found on a quick search: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843247/

What is your hypothesis and how did you plan to test it? It is possible to over-express a gene in E coli by inserting it into an expression plasmid and transfecting this into the bacteria. Some of the plasmids have 'switches' that can be used to turn on transcription and generate the cognate protein.

Get back to me with more details of what you are trying to do and i can advise you better.

Sybee

lpullela2424
Posts: 2
Joined: Sun Aug 27, 2017 12:08 pm
Occupation: Student

Re: How to increase gene expression of HSP in E.Coli bacteria

Postby lpullela2424 » Thu Aug 02, 2018 3:57 pm

Hi Sybee!
Thank you for your reply! I actually did more research on this topic and actually came to learn that introducing plasmids which translate proteins for eukaryotic genes is actually not that ideal in prokaryotic organisms such as E. Coli because the transfected genes undergo so many post-transcriptional modifications, and so most of the times it may not even work. I was trying to induce a beta-amyloid plasmid into the E.Coli and increase gene expression on the heat shocks (HSP are part of the E.Coli but not the amyloid protein). I have changed some aspects of my project to solve this issue. I will be doing everything in test tubes without using organisms; that way there is no chance of PTMs or mutations or anything! Thank you so much anyway for getting back to me!

SciB
Expert
Posts: 1852
Joined: Fri Feb 01, 2013 7:00 am
Occupation: Retired molecular biologist, university researcher and teacher

Re: How to increase gene expression of HSP in E.Coli bacteria

Postby SciB » Fri Aug 10, 2018 6:45 pm

You are most welcome!

Let me know if you have more questions on your project. It's always good to run your ideas by someone else just to get their feedback. As a scientist myself I always do this and have caught potential errors and avoided problems that way. Two or three or four heads are usually better than one.

Best wishes,

Sybee


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