Should I clone a gene with RBS found in upstream of the gene into pET28a(+) for expression in E. coli BL21(DE3) or I just need to clone only gene sequence started from ATG ???
Thank you for your help!
The RBS can have an effect on the translation efficiency and this might be important. Anyway, there is a strong RBS in pET28 so I would use this and clone the gene from the start codon.
Ideally, when cDNA is used, it is likely that no RBS is present in the gene so, vector backbone just after the promoter region, after transcription start site should have the RBS which generally is present in 5' UTR of the gene.
RBS containing the Shine-Dalgarno sequences are essential for interaction with 16s rRNA in 30S subunit of ribosome. This interaction is necessary for proper binding of ribosome and translation initiation.
Check the vector backbone whether it has the necessary sequences after the promoter which could be transcribed and hence help in translation of your protein of interest. Then you can use the cDNA only for cloning.
Only the gene is sufficient, although do consider the restriction sites flanking your gene of interest.
Abhishek
Thank you very much for your kind answers.
Actually, I have successfully expressed some other genes that I have not found any RBS in cDNA. But this time, I have been expressing 2 genes that RBS locates in the upstream of those with the similar conditions for cloning and expression. But I get nothing in SDS-PAGE, evenly insoluble part.
So I wonder about RBS in cDNA as I mentioned above.
Could you give me some advice?
Dear Nguyen,
Why don't you try leaving the RBS when inserting your gene. I mean, PCR your gene of interest downstream of the RBS and ligate that into your vector.
Abhishek
Ah, Actually I just amplified the only gene without RBS. I forgot to mention here.
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