Recently, I tried to transform a 100Kb gene cluster into industrial Streptomyces chassis like Streptomyces avermitilis SUKA22 or Streptomyces albus J1074 by conjugation, but failed. I had optimized the concentration of Mg2+, medium (MS, ISP4, 2CM), but no transformants were obtained. Does anyone have experince on transformation or heterologous expression of large gene clusters?
There are a lot of factors that can contribute to the success of a conjugation - as you've said the medium is a good place to start, but there are a few other things to think about.
The ratio of donor (conjugation appropriate E. coli strain e.g. S17.1) and recipient (Streptomyces spores) in the conjugation are important. On your plates do you see an overgrowth of E. coli or Strep after a few days? Sometimes less is more, and a fresh Streptomyces spore stock can help. Also take care with the timing of the heat shock step to inactivate restriction enzymes in the Streptomyces spore stock if relevant. Consider plating controls to ensure your spores survived the heat shock.
The timing of the antibiotic flooding step is also an important factor - overlay too early and you'll stop any growth coming through, too late and you may find a proliferation of you donor E. coli on your plates or conjugation negative Streptomyces. ~15 h seems to work for me, but wait until you can just start to see some growth before you add your antibiotics.
Also sometimes patience is the answer! A lot of protocols say it takes ~72 h for colonies to emerge, but I've seen them come through in less than two days, or sometimes after 5 or 6 days something will just pop up! Just make sure to re-streak and PCR/sequence to confirm it's got your desired cluster integrated.
Good luck!
Luke Stevenson Thanks for your suggestion. Recemtly, I have tried a procedure of conjugation referred to the paper "Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces". It did work in S. albus J1074.
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