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Suppose I have a hetrologous (eukaryotic, plant) gene sequence that I want to express in E Coli under the control of a regulable promoter (say the lac promoter induced by IPTG) how exactly does one add the promoter sequence upstream of the gene?

Are there any tools to do this?

e.g. Say this is the gene itself, what sequence modification is needed to put it under the control of the lac promoter? The goal is to induce overexpression of the protein coded for by the gene on addition of IPTG to the medium (if I understand the strategy correctly)

atggctaccg ataatgacag ctctgaaaac cgtcgtatgg gtaattacaa gccgtccatc       60

tggaactacg acttcctgca gtccctggct acccgccaca atatcatgga agagcgccac      120

ttgaaactgg cggagaaact gaaaggccag gtgaagttta tgtttggtgc cccgatggag      180
.................................................................
gagccgttcg tgacgttcaa cctgaacagc gttcgtggtt cccatttctt ttacgagttt     1560

ggtgacggtt tcggtgtgac gaatagctgg accaaggttg acatgaagag cgtcctgatt     1620

gatccgattc cactggatga agaataatga                                      1650
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If you're expressing the gene in E. coli, then you're almost certain to be using a plasmid or other ectopic gene vector. There are a wide variety of plasmids available, with many different promoters. With a commercial plasmid, you're not adding in the promoter sequence, but the gene sequence. You pick the promoter you want and then clone your gene sequence into the plasmid's multiple cloning site, which is designed to lie downstream the promoter.

There are a variety of tools to design and order plasmids. A common plasmid repository is addgene and my favorite tool for designing plasmids prior to ordering is APE.

Design the plasmid with APE or another tool, to be sure that everything looks good, then you can order the appropriate plasmid vector and enzyme cut site-flanked gene sequence and go to town cloning.

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  • $\begingroup$ I have asked a follow up question here & it would be great if you can help with any comments / advice: biology.stackexchange.com/questions/48451/… $\endgroup$ – curious_cat Jun 29 '16 at 8:05
  • $\begingroup$ I believe that @March Ho's comment to your follow-up question provides the best answer. I've never seen a 'promoter menu' offered. Rather, promoter sequences are generally specified within a given cloning vector and can otherwise be found using tools such as GenBank. Hope that helps. $\endgroup$ – Forest Jun 29 '16 at 16:11

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