Autoinduction media
Protein expressed well in E. Coli colonies on agar plates, but when I cultured those colonies, I got nothing???
2023.06.07 23:00 Academic_Wrangler_65 Protein expressed well in E. Coli colonies on agar plates, but when I cultured those colonies, I got nothing???
hi fellow lab rats,
thanks in advance for your help with this. i feel like i must be missing something obvious, so an outside perspective to my own will be very useful.
TLDR; my protein library expressed great in E. coli colonies on agar plates, but when i cultured these colonies to prep and test the protein variants, expression was so bad i got functionally no data. what could cause this?
full explanation:
i recently made a library of a red fluorescent protein (RFP), chemically transformed that library into T7 Express (a commercial line of highly chemically competent E. coli), and plated my transformants on agar plates with appropriate antibiotics. i grew these plates at room temp (just out on my lab bench) over this past weekend. on monday, the plates looked fabulous -- i could easily tell that my protein was expressed well because my colonies were very pink in normal lighting and fluoresced brightly under illumination by a ~500nm wavelength light box. given that my plates looked good, i picked single fluorescent colonies into wells of 96-deep-well culture blocks containing antibiotic autoinduction media, one colony (and therefore one protein variant) per well. i also picked a few wells of the library template into these blocks to use as a control. i then cultured these blocks at 30C for ~48hrs and shaking at ~300rpm. today (wednesday) i took them off the shaker to prep and test my protein variants to see if i got any hits, and noticed immediately that the bacteria pellets, including those expressing my control, were barely pink. i prepped the proteins anyway and got unusable data because they expressed so poorly.
i have been evolving this RFP for the past year; this is my tenth library of this RFP, and i use the same workflow described above for every library with few issues until now.
does anyone have any ideas as to why my protein would express well on agar plates but not in culture? i double checked that the autoinduction media i used was in-date, which it is. i also checked that the shaker was actually on before i left on monday, checked it again on tuesday, and it was still shaking this morning, so i don't think it was stopped at any point during the culture period.
my plan for now is to just re-plate the library (i always save the library DNA for this very reason), re-do the experiment, and hope for the best, but any advice or insight will be much appreciated. thank you!!
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2022.03.15 19:52 BiochemNerd22 Why do my cells only express one protein at a time?
I’m trying to co-express 3 proteins in BL21 Ecoli. Each of the 3 expresses fine on their own.
I put protein A in pET-21b and then protein B protein C are together in pACYC-duet. When I attempt to express them, I only see protein A, but not protein B or C.
If I try to express protein B and C together (but leave out the protein A plasmid) now I get robust expression of protein B but still nothing for protein C.
Does anyone know what could be causing this issue?
Notes: All 3 have the same T7 promoter with the lac operator and T7 terminator. They also have the same shine dalgarno element. I have tried LB, TB, and autoinduction media at room temp, 30C, and 37C at times 4hr-24hr. All 3 genes are codon optimized for ecoli (and they all express fine if alone in pET-21b).
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2015.01.19 23:52 SarahMackAttack SUMO vs. Trx tags for increasing solubility of proteins
I'm trying to purify a squid protein in BL21 E. Coli that refuses to solubilize. I've done regular IPTG induction and a couple types of autoinduction media, but it hasn't worked. I'm about to try a solubility tag and I'm wondering if anyone has had experience with both SUMO and Trx tags for solubilizing tricky proteins. From my reading it seems like both should work theoretically, I just want to see if someone had some hands on experience for this one. Any insight is appreciated!
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2012.06.27 14:53 mimac [R] A few papers
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http://rodzice.org/