I am transforming cyanobacteria with a vector containing antibiotic gene as well as my construct. However, after transformation the strain is growing in very high concentration of antibiotic suggesting the successful transformation if my vector but I failed to get any band of my construct. What could be the reason? How can I troubleshoot?
Hi,
sounds like re-ligated empty vector to me. did you try to isolate the plasmid and do a RE-digestion with the RE you used for cloning?
You could also use (colony)PCR to see if the fragment is in your vector.
You could have also coincidently transformed your cyanobacteria with the uncut plasmid.
good luck,
Mina
Did you insert the resistance to the antibiotic in a gene of cyanobacteria? In this case you can chrek by PCR, I assume you should have the primers corresponding to thecyanobacterial gene.
Which resistance cassette have you used for selection? We struggle with Chloramphenicol in Synechocystis.
Which cyanobacterium? Some are pretty good at developing spontaneous resistance (probably impaired uptake/enhanced efflux). Also, some cyanobacteria prefer single-homologous recombination rather than double-homologous recombination. My recommendation would be to retransform and check for presence of the desired genotype at an early stage, well before segregation.
I am using pSYN1 plasmid which has spectinomycin resistant gene. The strains I am using are local isolates of cyanobacteria. After transformation, my strain is growing very well in the even higher concentration of spectinomycin but when I did a PCR using construct primer, confirmational primers (Neutral site primers) as well as primers for spectinomycin resistance gene, I found no band.
Dear Ms. Shinjinee Sengupta,
pSYN1 is a Synechococcus PCC7942 vector, which incorporates your gene of interest in between neutral sites NS1a and NS1b, it might be possible that your cyanobacterial isolate does not have a sequence homologous to NS1 of 7942 in its genome.
Also, I would like to know if you have checked spectinomycin resistance of the wild type strain.
you can either identify neutral sites in your strain, or use a broad host range plasmid such as pRL1383a (based on RSF1010 plasmid), which is known to replicate in a wide range of cyanobacteria.
All the best.
https://www.addgene.org/70692/
Could try to know the spontaneous resistence of your local strain to spectinomycin? Several clones spectinomycin resistant should have obtained after transformation, did you try by PCR the presence of the sp resisitancein several clones,?
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