Difference between revisions of "Part:BBa K2014011"

Revision as of 12:15, 21 October 2016

pxylF-xylA->sfGFP_B

Usage and Biology

We designed pxylF-xylA->sfGFP_B as a fluorescent marker to measure to what extent codon optimization may change the rate of heterologous protein production in E. coli. It is believed that by codon optimization one can substantially increase the gene expression and that the optimized gene will more effectively compete for cell resources and will be more accurately translated [Kane JK, 1995]. We would like to check which approach to optimize a reading frame is the best and to what extent it can improve the expression of the optimized gene. We consider improvements of such traits like: codon usage, codon adaptation index, contexts of codons and secondary structures in coding sequences. We intentionally started our comparisons from implementing general optimization rules, which effects can be easily compared in simple induced expression experiments.
We have started from a simple optimization of sfGFP in which we changed every codon of sfGFP [Pedelacq JD, 2006] to the most abundant synonymous codon in all reading frames of E.coli K12 orfeome, according to the codon usage table generated for us by Prof. W. Karłowski. At the N-terminus of coding sequence there is a stable 6-histidine tag (Fig. 1). The reporter gene is cloned under a relatively weak xylose induced promoter - pxylF-xylA, which is a wild-type E. coli promoter.

Fig. 1. <b>Fig. 1. The scheme of the biobrick: BBa_K2014011. B letters correspond to the most frequent codons in E.coliK-12 orfeome.

Sequence and Features