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Are two promoters needed for co-expressing 2 proteins from the same plasmid in S


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#1 donny

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Posted 08 April 2013 - 08:56 PM

I'm working on Saccharmoyces cerevisiae for the first time and am intending to co-express 2 proteins from one plasmid. I have initially thought I can simply clone the genes serially and they will express under the same promoter, like in E. coli. Would this work in S. cerevisiae? I'm asking because I've read many papers and they all use pESC vector such that each gene is under the control of their own promoter. Is this essential?

#2 neuron

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Posted 10 April 2013 - 01:39 AM

Yes It is essential, In case of E.coli also, I think it is essential. For instance pETduet vector which is used to coexpress two genes in e.coli also has two separate promoters . Theoretically also if we see, translation would stop after reaching to stop codon, then to start the translation for second gene, where will polymerase bind?..there has to be some promoter sequence for that....I hope I am making sense (I may be wrong thoughPosted Image )

Edited by neuron, 10 April 2013 - 01:39 AM.


#3 donny

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Posted 10 April 2013 - 06:52 PM

I have cloned two genes in series using one promoter but with an RBS before each gene for co-expression in E. coli and it worked. I've been reading up a little and from what I understand, prokaryotic mRNAs can be polycistronic while eukaryotic mRNAs are only monocistronic. So, many proteins can be translated from a single mRNA in E.coli but only one protein is encoded in every mRNA in yeast.

The promoter sequence controls transcription, not translation. Translation is performed by ribosomes. It binds to the RBS on the mRNA and stops at the stop codon in the RNA. Transcription is performed by polymerases and stops at the termination sequence in the DNA. I hope I got that right. I'm more a chemist learning molecular biology on the fly.

#4 neuron

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Posted 11 April 2013 - 08:16 PM

I completely agree with you donny. I mixed translation and transcriptionPosted Image ...But my question is when we do cloning in an expression vector. we just put our DNA sequence ahead of promoter sequence, transcription and translation are taken care in bacteria. Therefore when you transform your vector into bacteria, it forms mRNA first and then DNA..so to make mRNA also promoter is required?????

#5 donny

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Posted 12 April 2013 - 08:14 AM

Firstly, we place the promoter in front ofs the DNA sequence of the gene, not the other way round. Promoter sequence is where the RNA polymerase binds so as to transcribe the gene sequence into mRNA. The mRNA is then translated into protein by ribosomes. So yes, promoter is required to make mRNA. That's my understanding. DNA replication is done by DNA polymerases and other proteins I believe and does not involve mRNA. It may involve RNA in retroviruses but that's way beyond of my knowledge.




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