Dear scientists,
Am working in pseudomonas bacteria, where i could find lot of resistant genes, but when i extract the same with plasmid DNA extraction kit, no dna could be extracted.
Is there any method to confirm a strain carrying plasmid? Like PCR Primers?
Since Pseudomonas could integrate any kind of plasmid genome into a chromosomal DNA.
I am bit confused whether -a strain carrying plasmid or the gene integrated at chromosomal level without a plasmid.
use a strain for plasmid extration and run the gel.
It is easy way to see.
Dear Dr. Mario Hirata,
I have done the same and checked it, there is no band i could see. sir.
I have the same problem, but you need to grow the bacterial and extract from a big amount, because can have a small amount , if is clinical samples in media with antibiotic that it is resistance.
to make PCR you need to have suspect of gene presence in the plasmid thar is not in genomic DNA. Did you search in the literature in your country any presence of plasmid?
Did you try to study resistance gene in genomic DNA? maybe the strains are resitance by mutation, that is commum in this species.
which drugs they are resistance?
Maybe I can help you looking for these mutation in genomic DNA
Dear Dr. Mario,
This strain is resistant to all drugs except imipenem and monobactam.
i cultured this and extracted from ample amount of inoculum.
Bioinformatica analysis revealed: It carries integron and blaVIM-2 genes which i come to know through NGS data analysis.
And multiple aminoglycoside resistant genes and genomic islands.
But my worry is ? is it possible to carry all these genes in chromosomally? or not?
Thanks for your supportive answer sir.
Good news that you sequenced by NGS. Now you need work in bioinformatic and try to compare with many plasmid that are described until now in the data bank.iF YOU See any sequency that you can suspect to be related to plasmid, you just have your answer.
I suggest to work hard in analyse this NGS rearching for these plasmids.
If you send this data to a good bioinfoatics, problably they will answer your question
best regards. If you want any help in this fields I have 2 pos doc that can help you to analyse this sequence.
Many natural plasmids are quite large and unless you take special precautions may not be easy to observe using a typical plasmid preparation. I agree with Mario that you should be able to learn what you need from your sequence data.
You can try to make PFGE (Pulsed Field Gel Electrophoresis) and treat the block with nuclease S-1, you will only get plasmid DNA, even in the case of large plasmids.
Dear Nandagopal, I am in general agreement with the advice offered above, and suspect that you should be able to determine whether it is likely that your strain is harbouring a plasmid from the NGS data - though you can never be too sure that it is not integrated into the chromosome. If you want to demonstrate that there is a freely-replicating plasmid, you will need to either undertake a lot of PCR reactions (targeting all of the plasmid in an over-lapping series of fragments; one or two of which will not amplify if it is integrated), or try to examine total DNA extracts for the presence of plasmid bands (which is difficult if the plasmid is large and low copy-number). However, experimentally it may be easy to demonstrate the presence of a self-tranferable plasmid by conjugation into another pseudomonad (P. putida KT2440 is often used) if the plasmid is carrying an antibiotic resistance gene. Regards, Andrew
Dear Dr. Andrew sir,
Thank you very much for your excellent explanation and answer. i agree with your suggestions and will take further sir.
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