Transformation gone wrong! Please help! - (Oct/03/2008 )

I recently set up an experiment to restore the biosynthetic pathway of pyocyanin production in two mutant P. aeruginosa strains; one mutant makes a red pigment, and the other makes a yellow pigment. The goal was to transform the mutants by geting them to take up a plasmid from E. coli that had the genes to restore the pyocyanin (blue pigment) pathway. In addition to the pathway genes, (phzM and phzS), the plasmid also had tetracyline resistance. The students purified the plasmids last week and froze them for one week; on Tuesday, they were given competent P. aeruginosa cells of both strains, which they transformed with their plasmids. The results: the mutants took up the tetracycline resistance, but not the pyocyanin pathway. This experiment has worked well in the past, but with this class, I was baffled! Does anyone have any suggestions as to what went wrong?

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IF you could extract plasmid from the pseudomonas transformant and check for presence of the gene that would be good.
I suspect something like an empty vector going in .
did u check the plasmid purified w/ RE prior to transformation into Pseudomonas???

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in addition to an empty vector, you need to make sure that the structure of the vector is correct (ie promoter, terminators) and that there are no sequence changes to the phzM and phzS genes, which may have become mutated.

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The students ran a sample of their purified plasmids on an agarose gel, confirming that they had gotten plasmids. If the vector was empty, then why did the mutants pick up the tet resistance? Is it possible that the plasmids were somehow corrupted?

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Do you think that its possible that the phzM and phzS genes could have become mutaed? If so, how? Do you think that the medium had anything to do with the results?

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By being empty i mean the vector doesnt' contain the phz and phs genes and having two genes being mutated at the same is highly unlikely. Just run a restriction digest to confirm the size. running a plasmid on gel could tell that ur extraction is working but not the actual size of the plasmid.

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It would depend on how the plasmid was constructed. If the plasmid were built by PCR, it could be PCR induced mutation. (ie student did not use a proof reading polymerase) And any plasmid that is amplified by E coli, is subjected to homologous recombination especially is the genes have highly similar to each other.

We really do need to known how this plasmid were built to guess at the cause.

In any case as Hanming86 has suggested, check the plasmid by restriction site mapping. The most likely cause is that the plasmid is blank... empty... ie it is the original vector that does not carry any insert.

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The plasmid was created as follows (from Mavrodi, et al. "Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PA01" in Journal of Bacteriology, November 2001):

P. aeruginosa PAO1 phzM
and phzS knockout mutants were generated by using a gene replacement strategy
previously described by Schweizer (50). To mutagenize phzM, the gene was
amplified by PCR with oligonucleotide primers METHYL1 and METHYL2
(Table 1) from cosmid 1G5 DNA of P. aeruginosa. The 1.26-kb PCR product was
digested with EcoRI and BamHI, cloned into pNOT19, and inactivated by
insertion of an 879-bp aacC1 cassette from pUCGM into a unique EcoRV site
(Fig. 1A). The resulting plasmid, pNOT-ORF1-Gm, was digested with NotI and
ligated with the 5.3-kb MOB3 sacB cassette (50).
To inactivate phzS, the gene was amplified from cosmid 1G5 target DNA with
oligonucleotide primers OXY1 and OXY2 (Table 1). The 1.24-kb PCR product
was digested with XbaI and SphI, gel purified, and cloned into pNOT-19. The
aacC1 cassette was then inserted into a unique ScaI site within phzS (Fig. 1A).
The resulting plasmid, pNOT-ORF2-Gm, was ligated to the MOB3 sacB cassette
and used for mutagenesis.
Plasmids containing the sacB cassette and the inactivated phzM or phzS gene
were mobilized in P. aeruginosa PAO1 from E. coli S17-1. Following selection for
double crossovers, isolates were screened by PCR for the presence of plasmidborne
bla and sacB genes in the genome. The -lactamase gene was detected by
PCR performed with primers BLA1 and BLA2 (Table 1), which amplified a
744-bp fragment of bla. The sacB cassette was detected by PCR performed with
primers SAC1 and SAC2 (Table 1), which amplified a 1.05-kb DNA fragment.
The cycling program included a 1-min initial denaturation step at 94°C, followed
by 25 cycles of 94°C for 45 s, 53°C for 45 s, and 72°C for 1.25 min. The presence
of mutated alleles of phzS and phzM was confirmed by PCR performed with
oligonucleotide primers MET1 and MET2 and oligonucleotide primers
ORF2UP and ORF2LOW (Table 1), respectively.

I received the E. coli with the pUCP-ms plasmid already inserted; I grew the E. coli in LB, and then froze samples in 20% glycerol at -80 degrees celsius. Because this I am the lab tech/teaching assistant for the lab, I was told by the instructor that the students would purify their own plasmid from the bacteria that I had frozen. That could be the first mistake. The reason I'm so puzzled is because last semester, I grew up the bacteria from a vial of frozen bacteria from the same stock supply that I used this term. I am going to try and run the experiment myself tomorrow and see if I can get better results. If it doesn't work, I'll run a digest and go forward from there. Thanks for the help!

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You might also check that the P. aeruginosa strain you are transforming into is the same as the one which worked before. I could easily imagine a strain difference making this fail.

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