Hi All,
I am currently doing recombineering to insert two mutations on a plasmid, but after successfully recombining the sequence, the cells from my plates don't grow on LB broth.
This is my workflow simplified:
1)Transform NEB10b with my plasmid (Amp resistance) then make them electrocompetent and electroporate Red/ET plasmid (Tet resistance).
2)Grow at 30degrees due to temperature sensitivity of Red/ET, induce Red/ET machinery by adding L-arabinose and shaking at 37degrees for 1 hour.
3)electroporate intermediate sequence for selection. This sequence confers plasmid with kanamycin resistance and streptomycin sensitivity. After electroporation, I shaked at 37 for three hours to allow recombination to occur.
4)Checked colonies by cPCR, had a mixed population of NEB10 with intermediate sequence and the old sequence that I want to replace.
5)Miniprepped and retransformed to separate the plasmids - done successfully and obtained colonies with just the kanamycin resistance and strep sensitivity sequence which grew in Kan+Amp plates.
6)Picked the colonies that showed more sensitivity to streptomycin and electroporated Red/ET plasmid. repeated step2.
7)electroporated final sequence with my two mutations, this sequence should replace the intermediate sequence. Allowed bacteria to shake at 37 for 3 hours for recombination to occur.
8)Plated on streptomycin and ampicillin, and got single colonies which I assayed by cPCR for the intermediate kanamycin resistance sequence as well as for the final sequence. Around 1/4 colonies had a mixture of recombineered and non-recombineered plasmids.
At this stage I should repeat step5 to separate this mixed plasmid population. But when I get the bacteria from the plate and put them in LB broth with ampicillin I get no growth. I tried shaking them for 2 days at 37 and nothing... I also added less ampicillin concentration (50ug/ml) and checked that the broth is okay by shaking another bacteria from a diff. experiment which did grow. The bacteria are also freshly plated on the previous plate but somehow they are dead??
Could it be something to do with the Red/ET? or the streptomycin? Any suggestions please!
Does anyone know what could be going on??
Thanks!
Could be that your final construct is toxic at a high copy number?
Have you tried growing them in SOC media?
Hello, maybe your recombinant lost lactose-consuming ability try different media for growing starting from the nutrient broth.
Complicated assay! The last step where you see live colonies is on strep & amp plates. Ampicillin resistance is notoriously "leaky" since the beta-lactamase is excreted into the medium.
Do your colonies grow under just strep selection? What about on plain LB?
Hi everyone, thank you so much for your answers!
Robert Adolf Brinzer - The final construct shouldn't be toxic, but some unwanted recombinations could potentially alter the plasmid so that it loses functions, but I struggle to understand why it grew in the first place then. I will try to grow them in SOC media with some ampicillin if this could help.
Katie A S Burnette - I haven't tried growing them just under strep, but I will if you think this could help? I tried plain LB and got colonies, I then inoculated some of the colonies from the plain LB into increasing concentrations of LB with ampicillin and they grew!
- Badges
- Science topic