I've been trying to transform my plasmid conferring spectinomycin resistance into EHA105 (via freeze-thaw method). Unfortunately, I've been observing bacterial growth on the entire plate and not getting single colonies. I have tried to increase the concentration of spectinomycin, reduced the recovery time, and diluted the recovered cells before plating - same results. I am now planning to reduce the initial volume of the competent cells that I will transform (from 150 uL to around 35 uL).
1. What is the result of your baseline antimicrobial susceptibility testing (pre-transformation) with your strain for spectinomycin? Did you observe full bacteriostatic activity relative to reference for the bacterial species or, better, strain?
2. Is your spectinomycin stock intact/optimal/working/unexpired? What range of working doses have you used so far?
Consider the likelihood of spontaneous resistance prior to transformation, or the use of a misclassified strain (one that is known to be inherently resistant to spectinomycin). You can try running experiments to check for genotypic or phenotypic (enzyme activity, perhaps, beyond mere antimicrobial susceptibility testing) evidence of resistance.
Hi Aedrian Abrilla
1. We observed bacterial growth even in our negative controls (no inserted plasmid). We are actually trying to find literatures to confirm if this particular strain that we are working on is incompatible with our selection marker. Unfortunately, we cannot find any supporting paper, which is why we tried different ways to make it work.
2. We prepared/used new stocks of our antibiotics. So far, we've applied 50-200 ug/ml.
Thank you for your answers and suggestions! We will consider these.
I did some literature digging and apparently part of EHA105's construction involved transforming it with then curing it of a plasmid called pPH1JI. pPH1JI contains multiple drug resistances on what is probably a composite transposon, including resistance to spectinomycin & gentamicin. Only loss of gentamicin resistance was verified, so my speculation is that some other resistance gene(s) could have transposed to either pEHA105 or into the chromosome unnoticed.
That's assuming the strain you have is actually EHA105 though.
(Attached are the papers I got the info from)
Lawrence Yves Cheng Uy That paper talks about EHA101, the precursor strain to EHA105. The process of constructing EHA105 cured it of kanamycin resistance, and I don't think EHA105 existed at the time that paper was published.
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