I am trying to transfer a 70,000bp secondary-metabolite-producing piece of DNA from E coli. 12567 cells into Stretpomyces Albus and Amycolatopsis Balhimycina by mating/conjugating them on an ISP4 plate.
My protocol goes like this.
1) From an O/N culture, start a seed culture and grow till OD=600
2) spin down 5ml of culture per mating, wash with cold LB 2 times, and resuspend the pellet in 1/10th the original volume.
3) Add strep (or amycolatopsis) to 0.5ml TSB and heat shock at 50oC for 10 mins
4) Add 0.5ml of concentrated E. coli mix to each
5) centrifuge for 1:30, pour out most of supernatant, resuspend pellet in remaining supernatant and plate on ISP4 plus 10mmol MgCl2 plate (with no antibiotics)
6) grow in 30oC fridge, and 14-16 hours later overlay with 1.5mg/plate of naladixic acid and 3mg/plate apramycin.
As my control, I am, in addition to the 70,000bp DNA+vector, mating the vector alone into the strep. The strep is growing in A. balhymicina with about 30 colonies per plate (S. albus isn't growing, probably because the stock solution is dead), but the 70,000bp DNA fragment is not growing.
I've done the mating twice now, and gotten the same results.
My guess is that the size of the fragment is hindering the mating for some reason, but I'm not sure how or how to fix it.
If anyone has any suggestions as to how I can improve my experiment they would be greatly appreciated.
Hi,
i would remove the step with heatschok; use only 750 microgram of apramycin per plate and substitute nalidixic acid with fosfomycin. The fragment size does not influence the conjugation frequency in case of S.albus as a recipient.
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