I have two constructs of arabidopsis genes cloned in pet28 vector, transformed in rosetta. The glycerol stocks made three years back were streaked in kan plates, single colony checked with pcr and confirmed and induced using general protocol ( primary culture of 5ml, secondary culture of 5ml-OD 0.8, induction of 4ml secondary culture with 1mM IPTG for 4hours at 37°C, sonication in 1X PBS, sample processing with loading dye at 100°C for 5min, ice for 5min and loading in PAGE gel). Unfortunately the profile of induced and uninduced are exactly same as run in gel. The person who had cloned it 3 years back was able to induce the protein in the supernatent with the very same conditions. Can anyone point out what wrong could I be doing? I'm attaching a gel image representative of the kind of result that I'm seeing.
Thanks a ton.
Did you add chloramphenicol to Rosetta cells to your cultures, as well? The plasmid in Rosetta cells that encodes for rare tRNAs is chloramphenicol-resistant: https://www.emdmillipore.com/US/en/product/RosettaDE3-Competent-Cells-Novagen,EMD_BIO-70954. To really get the protein expression benefits of Rosetta cells, it looks like you'll need to select for chloramphenicol. Your sequence likely has rare codons that E. Coli has trouble with, so expressing in Rosetta without chloramphenicol selection is analogous to expressing in regular old BL21s. I would repeat everything, but also with addition of chloramphenicol (and with kanamycin) to your primary and secondary cultures at 34 ug/mL final concentrations. (To keep is easy for large-scale expressions, make 1000x chloramphenicol stock in >70% EtOH, 90% to 95% is what I'd recommend.) Let me know how that works, if that doesn't do it, then you may have to tweak induction time and temperature, IPTG concentration, primary culture growth, etc. Good luck. -Brian
@Brian no I hadn't added chloramphenicol! Thanks for the suggestion. Was the first time I actually worked with Rosetta so I treated it like how I treat BL21 DE3.
No problem, glad to help. Roughly half of my expressions are in Rosetta cells nowadays, and chloramphenicol selection is necessary. If you have to re-transform, you don't need to select for Kan+chloramphenicol in your transformation. (I plate colonies on LB+whatever antibiotic my plasmid is resistant for.) But, you do want to add Chloramphenicol to all of your primary/secondary cultures. Other that that, everything is the same. Should be fine.
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