I seek for advice on the next point:
I need to coexpress two proteins from two independent plasmids, normaly in BL21(DE3). The first one would be a pET15b or pET26b-like one (pBR322 ori). Induction would be triggered with IPTG. For the second one, I would need a system permitting to easily tune expressed amounts. Idealy, I need to express lower and higher amounts than those of the first protein. I am aware this will be dependent on many parameters (e.g. protein sequences, or that p15A copy numbers are usualy half than for pET vectors). To simplify the discussion, let us consider a case in which the two proteins would be the same one.
I am considering the possibiity to use for the second protein a system controled by arabinose like the one on pG-KJE8 from Takara (pA15 ori, CmR, coding for AraC, I would remove before the Pzt-1/TetR/GroES/GroEL part). In your experience, do you think it is going to be possible to do what I want by playing with IPTG and arabinose amounts, or it is going to be complicated by other things (I read for instance on cellular heterogeneity with arabinose induction)? Would you advice another type of approach?
Thank you very much in advance,
Hello, see link below may be useful for you. Good luck!
https://www.researchgate.net/publication/11830922_Novel_Vectors_for_Co-Expression_of_Two_Proteins_in_E_coli
there are a variety of options and arabinose is certainly one of them. The problem you might have is that T7 expression is very high level and you will never match it with the araBAD promoter, so it might be quite difficult to get higher expression. There are a variety of other regulated promoters as well for E. coli. You can get around the heterogeneity problem by using the right strains otherwise it will be an issue.
You might take advantage of the Ribotite system (See Article Dual transcriptional- Translational cascade permits cellular...
), which seems to offer tuneable expression and better cell-to-cell homogeneity than the Ara system. Construct a bicistronic T7 expression vector where the tuneable gene is under control of the Add-A riboswitch, and control the expression level of the invariant gene by designing a proper RBS using e.g. the RBS calculator of the Salis group.Hope it helps!
Hello, see link below may be useful for you. Good luck!
https://www.researchgate.net/publication/11830922_Novel_Vectors_for_Co-Expression_of_Two_Proteins_in_E_coli
First of all, I really thank you all very much for your advice.
After discussing with other people, I understand that the pBAD promoter is not going to provide same levels of transcription as the T7 (as Michael highlighted). Moreover, it seems there are no promoters of comparable strength to T7?
I therefore forget about the idea of controlling relative expression levels with molecular inductors such as IPTG or arabinose. What Alejandro is suggesting is going to be hard to apply to my case. To screen different relative expressions, I would need to build combinations with different RBSs, which should be tested separatedly. This would be feasible for studying a few protein cases. However, I did not mention that using two plasmids was imposed by the fact that we want to screen couple combinations by crossing two libraries of sequences. Working with separated plasmids reduces the biology molecular work. For the same reason, the experimental task would increase enormously if in addition I had to build each library with variable RBS. I don't think that we can correctly predict expression levels for a given sequence.
Unless you added some other commentary to correct my view, I will clone my sequences in two T7 vectors and I will cross fingers. I will use pACYC/pDUET or similar, which are also similar to those proposed by Karen.
Again, thank you very much for your help
I might suggest you consider using lower expression promoters and not T7 for the experiments you are proposing. The T7 system is great when you are trying to massively overexpress a protein, but it is not physiological in any sense of the term. If you are looking at protein:protein interactions you might find it more reliable to have both your plasmids using moderate strength promoters instead of T7.
I absolutely agree with you, Michael. The major problem is the sensitivity of the high-throughput "reading" assay, which limits in our hands the use of "low expression" situations...
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