I would like to test functional activity of the cDNA from the transcriptome from a pigmented zooplankter in a modified K-12 E. coli strain which can produce the same pigment. However I am not experienced in cloning and thus looking for a simple vector to transform the strain, which I can use for both, cloning (EST sequencing) and expression. I could use the ZAP lambda system but it is quite expensive and involves a bunch of intermediate steps like blue-white screening.
I first thought that the pJET1.2 plasmid would be ideal. This vector should accept up to 10kb, has a lethal gene for positive selection, and a T7 promoter. I have read here on the researchgate a suggestion to use the bacteriophage CE6 (Novagene) to provide the T7 polymerase to a K-12 strain lacking this enzyme. Has someone really succeed in doing so? Is there any other positive selection expression plasmids which are as simple to use as the pJET? Could the pUCXKT plasmid (Prosser et al. 2015 in Biotechnol Lett) be a good choice?
I would be thankful for any help
I don't know pUCXKT so will refrain from commenting on it. As for pJET2.1 it is doable but there is a luck factor.
This pJET2.1 system is realy nice due to the zero background. But like many PCR cloning vector systems there no directionality. This mean your EST could fall under the T7 promotor or under Pluc-UV promoter, with 50/50 chance. Both should give some expression even under K12 strains but the T7 will be low. Furthermore either way the promoter will be rather far from the start codon (~300 bp).
One way to solve the T7 issue is to use BL21(DE3) [E. coli B] rather then K12. Here you induce the T7 polymerase by IPTG, also good to induce the Plac-uv5 promoter.
Putting theory aside, personally I did get expression from the pJET2.1 (I cloned a lipase) in DH5a (K12). It was enough to see activity but not for over-expression.
Another system to look at, although more expensive, is the pTOPO (Thermo). These systems are great for PCR cloning, especialy if you have many ESTs but tend to be expensive, at least in Israel.
Yoram
Thank you Yoram!
I checked pTOPO. It is also expensive here in Germany. I also have to stick to "my" K-12 strain because the strain express pigment, I need for testing the transcriptome (to fish for genes causing pigmentation plasticity in my organism of interest). Your experience with DH5a sounds very promissing to me: I do not need an over-expression (at least I think so) as I am mostly interested in the interaction of the insert product with the pigmentation pathway in the bacteria. EST sequencing is kinda side dish.
Sergej
If you have a simple assay it might work, did for me... For lipase activity we used Olive oil-Rhodamin B agar so we could look directly and pick active colonies. No IPTG needed.
Yoram
I see, I will have to play with the strain & plasmid, but it will be fun :) As my K-12 strain (BW-ASTA) is a derivate from BW25113, I could also try a pBAD33 plasmid and the arabinose promoter, will need cm selection then. But because I am new to cloning, I might by wrong.
I would have thought will also work with DH5a, and I love the Pbad promoter, but why is it better the pJET2.1?
The pJET2.1 give you (near) zero background due to Eco47IR gene that is toxic unless cloned, it comes pre-cutif you buy the kit, and atleast for me worked well.
If you are going for directional cloning then pBAD33 or one of the pET plasmids make sense, but its not simple PCR product cloning anymore. If you do wish to take this approach look at Gibson assembly. You will need to redesign your primers though.
Good luck
Yoram
https://en.wikipedia.org/wiki/Gibson_assembly
Thank you for the advice! I will first try the pJET out of the reasons you mentioned.
Kind regards
Sergej
With pleasure! You are welcome to write if you need further assistance or to tell us if it worked...
Yoram
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