I transformed a purified 11Kb plasmid (via plasmid mini prep) from an E.coli recombinant strain into the target host via electroporation. I got colonies but there was no DNA band during colony PCR. I suspected possible unusual recombination event as two of my target genes are flanked by two promoters that are closely homologous to the native promoter in the chromosome of the target host.
I misinterpreted a paper about R/M system, DNA methylation and electroporation efficiency as something to do with recombination (http://ieeexplore.ieee.org/stamp/stamp.jsp?arnumber=5517786&tag=1). So what I did is I transformed the plasmid into E.coli JM110 via heat-shock, did plasmid mini prep and transformed it back to the target host via electroporation. I did colony PCR and all of the colonies I screened went back positive.
I cannot find any factor so far that I did differently other than using the dam- dcm- strain E.coli JM11O which does not methylates the plasmid DNA compared to E.coli dh5alpha.
Im beginning to question if there is a possibility that a bacteria can aquire the antibiotic resistance gene during electroporation? Which should explain why I was able to see colonies in an agar plate with the antibiotic selective marker. And is there a possibility that maybe this is more apparent because the plasmid size is too big and hence more unstable.
Your question is a bit all over the place and unclear - I agree with Prashant that you should provide more details. However, your statement "I got colonies but there was no DNA band during colony PCR" makes we wonder if your PCR screen simply failed - its not uncommon that colony PCRs give false-negative results.
Dear Rey Sanjorjo,
Your situation seems a bit complicated I am not able to understand it correctly.
It would be good if you can explain your process using following parameters:
Which vector have you used?
What is your target host?
Which antibiotic resistance have you used?
Have you also checked restriction digestion with your E.coli colonies?
It would be easier to discuss further after knowing all the parameters.
Thanking you
Your question is a bit all over the place and unclear - I agree with Prashant that you should provide more details. However, your statement "I got colonies but there was no DNA band during colony PCR" makes we wonder if your PCR screen simply failed - its not uncommon that colony PCRs give false-negative results.
What Alexander said, colony PCR is super dirty and failure-prone.
I agree your question is not clear. It is good to clarify your points in the question to get relevant answers, comments or suggestions to fix your problems.
What did you detect from your colony PCR? The whole plasmid backbone or A specific gene for expressing specific proteins? If you are attempting to detect an insert from your plasmid using insert-specific primers by colony PCR, you will a chance to screen negative clones (or colonies that carry the empty plasmid without an insert).
Hello everyone,
It is a bit tricky to divulge some of the specifics about this as this is part of a patented project I am working with.
The host is Methylobacterium spp. and the plasmid contains a pmaxF promoter which is the strongest promoter used in prokaryotic methylotrophs. However the genome of Methylobacterium spp. contains a pmaxF promoter that regulates the expression of the genes associated with the methanol assimilation pathway.
My plasmid construct is
[pmaxF-(EcoRI-XbaI or EX)Gene1-Gene2-pmaxF-Gene3-Gene-4-Gene5(SpeI-PstI or SP)-Kanamycin resistance-OriT-TraJ-OriV]. The X and S sites have complementary overhangs and can stick together.
I constructed this plasmid via biobrick strategy using EcoRI-XbaI-SpeI-PstI cloning sites. All of the genes are heterologous while Gene 1,2,3 are codon optimized for Methylobacterium spp.
Protocol
1. Digested empty vector with E-X-S-P sites with E-P
2. Digested PCR amplified (E-X)pmaxF(S-P) band with E-S
3. Digested [pmaxF-(E-X)Gene3-Gene4-Gene5(S-P)] with XP
4. Ligated all 3 DNA to form [pmaxF-(E-X)pmaxF-Gene3-Gene4-Gene5(S-P)]
5. Digested the aforementioned plasmid with E-P
6. PCR amplified (EX)-pmaxF-Gene3-Gene4-Gene5(SP) using pmaxF forward primer with EX sites and Gene5 reverse primer with SP sites. I did this as the one who designed the primer designed the primer where dh5a can methylate, blocking XbaI restriction.
7. Digested the PCR amplified band with SP and ligated it to a previously digested
pmaxF-(EX)Gene1-Gene2(SP) plasmid forming pmaxF-(EX)Gene1-Gene2-pmaxF-Gene3-Gene4-Gene5(SP) plasmid.
8. Transformed to E.coli dh5a
9. Did colony PCR to detect positive colonies using Gene4-5
10. Inoculated positive colonies, plasmid mini-prep and did restriction mapping to confirm construct
10. Plasmid mini-prep the E.coli dh5a containing the final verified plasmid construct and transformed isolated plasmid to Methylobacterium spp via electroporation.
11. Did colony PCR and cannot find Gene3,4,5 (I have no primers for Gene 1-2)
12. So I used the isolated plasmid from E.coli dh5a to transform it to E.coli JM110
13. Did colony PCR
14. Inoculated positive colonies and did plasmid mini-prep, checked plasmid size
15. The isolated plasmid is then transformed into Methylobacterium spp.
16. Did colony PCR using Gene 3 for detection (almost running out of Gene4-5 primers)
17. I got 8/8 positive colonies for Methylobacterium spp containing final plasmid construct
The problem here is that we have not developed a way yet to plasmid mini-prep our recombinant Methylobacterium spp because the extracellular polysaccharide of Methylobacterium spp. elutes along with the DNA, contaminating it and making it impossible to see the plasmid size in agarose gel electrophoresis.
I originally thought it was a recombination action as there are two pmaxF that flanks Gene1-2, and these pmaxF promoter might (No genomic data for the specific methylobacterium we are using) be highly homologous to the one in the genome. But my Prof pointed out that methylation cannot affect recombination and I cannot find evidence in literature with my hypothesis hence my reasoning isnt true.
The Methylobacterium spp. we are using shouldnt be able to grow in kanamycin at 50ppm without the Km resistance gene. Is there a possibility that some of the empty vector must have cotransformed with the final construct contaminating my purified plasmid from plasmid mini-prep? Maybe that will explain why the bacteria was able to grow in a Km plate and why there was no Gene4,5 PCR band. I also think that because of the plasmid size, the empty vector which is only at 6kb must have higher transformation efficiency than the 11kb plasmid. And it was only possible to transform the 11kb plasmid when I got it from a dam- dcm- negative strain, wherein based on the attached journal can increase transformation efficiency.
To address the polysaccharide issue, we used to add a wash step to our Agrobacterium cells for minipreps. For 1.5 ml of spun down cells (overnight culture), resuspend in 1 ml of 'Agro-wash': 0.5 M NaCl, 50 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0 plus 5 ul 10% sodium sarkosyl, incubate on ice 5 minutes, spin again, aspirate supernatant (with drawn-out pasteur pipette) and proceed with miniprep. Hope that helps.
Hello Dr. Hayman, do you have the chemical components of the agro wash?
They are listed in my comment above. 1 ml of 'Agro-wash': 0.5 M NaCl, 50 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0 plus 5 ul 10% sodium sarkosyl. I couldn't find this in any of our papers, so found the recipe on the internet from a lab that said they got it from our lab (S K Farrand). My recollection is that we put the sarkosyl in the buffer, not adding it separately and I don't remember an ice incubation, but I passed on their recipe/procedure.
Im sorry, I missed the colon part, I thought it was a separate component. Ill try to find sarkosyl in the lab. I have already tried most of the components of this agrowash except sarkosyl along with acetone washing, boiling and not boiling.
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