![](https://parts.igem.org/images/partbypart/icon_generator.png)
Part:BBa_K2014009
pxylS-E1_5'UTR->sfGFP_B
Usage and Biology
We designed pxylS-E1_5’UTR->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 strong xylose induced promoter - pxylS-E1_5’UTR, which is shorter and has exchanged the native downstream 5’UTR comparing to the wild-type pxylF-xylA from E. coli .
We have compared the translational efficiency of sfGFP_B ORF with its non-optimized ORF (BBa_K2014002) and its inversely optimized form – sfGFP_W (BBa_K2014010) by measuring the fluorescence intensity of sfGFPs encoded by three different ORFs, which are under control of an identical promoter with an identical 5’UTR. Shortly, we compared the expression of sfGFP from three biobricks: BBa_K2014002, BBaK2014009, and BBa_K2014010 in E. coli DH5α cells grown in two rich media, LB and SB-PKB and in M9 minimal medium upon induction with xylose (0,4% final concentration).
Our results (Fig. 2.) indicate that in E. coli cells growing in rich media the difference in translation rate between reading frames composed of the most common and the rarest codons increases with the promoter strength.
In M9 minimal medium the codon optimization based on codon usage seems to be more important than in rich media. It is likely that in E. coli cells grown in minimal media tRNA pool and translational apparatus cannot be efficiently adjusted to translate mRNA molecules with reading frames composed of rare codons.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
None |