Description: Production of recombinant proteins in Escherichia coli systems is very attractive and popular due to its rapidity, low cost and well-characterized genetics. The most popular is pET (Merck, Novagen) lactose/IPTG induced T7 RNA polymerase dependent expression system, however this system leaks and introduces more mistakes in transcribed sequence comparing to E.coli RNA polymerase. That’s why we are looking for tightly regulated promoters, which can be induced independently of each other in one cell by sugars: arabinose, rhamnose, xylose or melibiose. We are trying to reduce their size to obtain minimal but fully functional promoters. Natural 5’UTRs of promoters that play role in translation initiation are substituted by other sequences. The modifications include better RBS positioning, introducing translation enhancers and removal of potential inhibitory secondary structures, which could interfere with translational machinery and decrease protein expression. To further evaluate translational efficiency, we also focused on codon optimization since it is believed that the frequency of particular codons in the gene of interest can cause expression problems because of rarely occurring tRNAs. According to bioinformatics analysis of codon frequency in E. coli we created two ORFs variants of sfGFP and sfRFP that are composed exclusively of the most frequently codons or the rarest ones.
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Year: 2016Visit Wiki
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Updated at: 8/9/16