Hi everyone,
Here we have a question regarding how to select a correct terminator for gene expression minoring in P. aeruginosa PAo1. Please let me know if you have any suggestions or ideas. Thanks a lot!
We are planning to put several promoter regions + reporter genes in a plasmid and then insert this plasmid into PAO1 to detect the gene expression of this strain under different environmental conditions. I am interested in genes A, B, and C from PAO1 and I want to construct a plasmid that contains the promoters of these genes + reporter genes (prompter of gene A + reporter 1------promoter of gene B + reporter 2-----promoter of gene C + reporter 3). Our method is similar to this paper: http://www.ncbi.nlm.nih.gov/pubmed/20646328.
After each reporter gene, we are going to put a terminator to stop the expression. What is the principles for selecting a terminator? Should I choose three different terminators, or I can just use a universal terminator for all the regions? Also, where can I get the sequences of the terminators? I noticed that there are several on-line tools that can be used to PREDICT a terminator based on a DNA sequence. However, how can we know the PREDICTED terminators will work well in the real world (in the plsmid or the bacterium that I will use)?
Please let me know if anyone have any ideas. Thanks!
I suppose you won't really know predicted terminators are working until you screen for the gene product. Since this is a cloning experiment, it might be reasonable to make a few different terminator sequences to try out. As you mentioned, I'm sure there are a few bioinformatic tools that could identify the common sequence among genes. An easy way to detect if a terminator is working effectively could be to make a tag after the terminator (FLAG, V5, etc.) and see if you can western probe for the tag; compare it to the same construct without the predicted terminator.
I prefer using different terminators since I'm thinking about homologous recombination.
If your concern is termination of translation you may embed double stop codon at the end of each GOI and a few hundred nucleotides as the spacer between each cassette.
You can find a transcription terminator database at "WebGeSTer DB" (http://pallab.serc.iisc.ernet.in/gester/).
WebGesTer Database (DB) is the largest compilation of intrinsic terminators of transcription. It comprises of >2,200,000 bacterial terminators identified from a total of 2036 chromosomes and 1508 plasmids.
To Ryan,
Thanks a lot for your help! That is a good idea. But first, we want to know which terminators we should test. We know that there are several good terminators in E. coli that are experimentally confirmed. Can we use these terminators in PAO1? In other words, if a terminator works fine in E. coli, will it works fine in PAO1. Alternatively, we can use predicted terminators from PAO1. But there are many predicted promoters in PAO! and it is hard for us to choose one.
On the otherhand, we do not have a device to do the Western Blotting here. Do we have an alternative?
Thanks again for your helpful advice!
To Saeid,
Thanks for the information for the database. Do you know that the terminators listed there are experimentally confirmed or just predicted?
Best wishes!
Terminators of ribosomal genes are usually very efficient and widely used in synthetic biology.
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