Transposase contamination in cloning - (Feb/16/2011 )

Dear all
I cloned a 1000bp human DNA in the pHIS1525 vector for expression in Bacillus Megaterium (from Mobitec).
As template for PCR I used another vector containing my DNA, I inserted my sequence directed, by NarI /EagI digestion of vector and insert. My cloning host was TOP10 (Invitrogen), which I use since a long time.
I got several clones and screened quite a lot of them.
Sequencing revealed:
I) clones that got my sequence in the right place on the vector with single base mistakes (which is normal at 1000bp)
II) clones that got 5´end of vector, NarI site and then (according to BLAST) a transposase sequence (IS10)
III) clones that got my insert sequence correctly in between the restriction sites, but instead of the 5´vector sequence a transoposase sequence (IS5)
I have done quite a lot of cloning so far and never have seen that before.
Does anybody have an explanation / experienced something similar ?
Please let me know.
pET

Top10 is closly releated to DH10ß ...as far as i know. DH10ß has been sequenced some years ago (see this link, just read the abstract: 13.5-fold higher mutation rate than MG1655) ...and what was seen is that i contains a lot more mobile elements than e.g. MG1655 ...therefore to my point of view it is not the best choice for cloning (although it is heavily used by people for whatever reason).

Structural plasmid instability can be an issue under certain circumstances ...it seems that you had the bad luck that your cloned DNA contains an hot spot for transposition of IS10. Normally, transposons get active if the cells are stressed ...so maybe aeration in your shake flasks was bad ...or something similar that motivated the IS10 to hop in your plasmid. If you are intersted see that link.

What you observed is biology in action ...as long as you have correct clones everything is alright ...but i would no longer amplify your plasmid in TOP10. There are minimal E. coli strains available that have all the mobile elements deleted (and a lot of other genes as well). See this link if you are intersted. I would try to change the strain that you use for cloning ...i use JM109 and for me it is fine.

But it seems that your gene that you are trying to clone is somehow toxic to the cells so that there is selection pressure to inactivate it (is there a bacterial promoter in front of that gene?) ...or that it contains a high degree of repeat sequences. Maybe you can check that by sequence analysis.

If you have any more questions i will try to answer them!

Regards,
p

pET on Wed Feb 16 14:51:28 2011 said:

Thank you pDNA for your quick and comprehensive answer.
I will definitively say good bye to TOP10.
However your reply roused some new worries:
1) How can I ever be sure to have no transposase problem in my clone, even if I get a correct result from sequencing, what if for example 90% of the plasmids in my clone are ok, but 10% have transposase (- which has due to minority maybe no influence on the sequencing result), and on further transformation of my plasmid prep in the expression host, I have 10% chance to transform the transposase ?
2) Can the mobile elements of the cloning host theoretically go on attacking my correct plasmid in glycerol stock, at regrowth of the clone etc., which means can I ever be actually sure about my clones ?
Greetings
pET

you can never be sure in biology

i would not panic ...for sure it is possible that by freeze-thawing you motivate your IS-element again to hop.

I would assume that the size of your plasmid is changed after transposition ...therefore you can easily check if something has happend to your culture by doing plasmid isolation and a restriction digest.

Are you going to do protein expression in E. coli with your clones?

Regards,
p

pET on Fri Feb 18 10:14:07 2011 said:

I did colony PCR and restriction digest before I sent my clone for sequencing and the mean thing was, that I got right size amplificate and digestion product as transposase was obviously the same size as my insert.
What do you think about the sequencing question (point 1 in my last post) ?
I want to do protein expression in Bacillus Megaterium if I ever get the cloning done.

Another weird lab problem from my repertoire with a completely different clone, but seeming somehow similar to me now.
I also then cloned human DNA (into pET30) first into TOP10, after verification into BL21, I got good protein expression (protein was insoluble).
After some month I reactivated the clone from glycerol stock and there was no more protein expression, PCR, restriction digest revealed that the plasmid was still ok, I tried to modulate expression, even transformed the plasmid from the old prep (I still had) newly into BL21 and did not got the protein to be expressed anymore. What do you think is the problem here ?
Greetings
pET

dear pET,

what plasmid backbone do you use? ...are your plasmids large in size?

Here is another link to a paper that was published in Microbial Cell Factories some months ago ...that deals more or less with the things you describe.

Sorry for that short answer but i catched the flu and do not feel realy well at the moment ...i will come back to you as soon as i fell better

Regards,
p

Dear pDNA

Hope you already feel better, I come back to your questions a bit late, because I also had the flu
The plasmid I use is pHis1525 (for protein expression in B.Megaterium), its size is 7474bp.
By the way thank you for the interesting link, it always is kind of reassuring to know also others have to fight these problems.
I still would be very interested on your comment to my previous questions.
Greetings
pET

pDNA on Fri Feb 18 15:25:38 2011 said:

dear pET,

what plasmid backbone do you use? ...are your plasmids large in size?

Here is another link to a paper that was published in Microbial Cell Factories some months ago ...that deals more or less with the things you describe.

Sorry for that short answer but i catched the flu and do not feel realy well at the moment ...i will come back to you as soon as i fell better

Regards,
p

so we are both back on track and healthy ...thats good!

Concerning your problem you described:
you wrote that both PCR and restriction digest pointed out that your plasmid is intact ...but did you sequenced it?
there is also the possibility that your T7 promoter gots inactivated by mutations ...when your gene product really is toxic there is strong selection in that direction ...that could inactivate your culture within 1 overnight culture. There was an publication on that topic as well ...but i'm currently not able to find it. If i do so i will post that link.

Another possibility is that the T7 polymerase gets mutated ...this was observed as well, see that link (although i did not really like that publication since it does not answer many questions).

So if your problem is really the toxicity of your proteins you should watch out for very leak proof systems like the strains that contain pLysS or pLysE and leak proof pET vectors (like pET30a). What vectors do you use at the moment?

Good luck!

Regards,
p