Part:BBa_K2014006

AraC-pBAD->sfGFP_W

Usage and Biology

We constructed AraC-pBAD->sfGFP_B as a fluorescent marker to measure the effect, codon optimization may have on 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 least frequent synonymous codon in all reading frames of E.coli K12 orfeome, according to the codon usage table generated for us by Prof. W. Karłowski. Thus, in sfGFP-W coding sequence appeared 7 dispersed AGG codons, 10 isoleucine ATA codons, 15 leucine CTA codons, and two pairs of consecutive CTA codons (these codons represent 40% of all codons in coding sequence). While designing the ORF the priority was to select the rarest codons. As a result, even though it does not meet the iGEM requirements, due to having a restriction site, we decided not to change the sequence. At the N-terminus of coding sequence there is a stable 6-histidine tag (Fig. 1). The reporter gene is cloned under arabinose promoter (AraC-pBAD) a wild-type E. coli promoter that is tightly controlled by L-arabinose and is used in pBAD expression vectors (Invitrogen, Thermo-Fischer).

Fig. 1. The scheme of the biobrick: BBa_K2014006. “W” letters correspond to the least frequent codons in E. coli K-12 orfeome.

We have compared the translational efficiency of sfGFP_W ORF with its non-optimized ORF (Ba_K1481002- provided to iGEM in 2014 by Poznan_Bioinf team) and its optimized form – sfGFP_B (BBa_K2014005) 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_K2014005, BBaK2014006, and Ba_K1481002 in E. coli DH5α cells grown in two rich media, LB and SB-PKB and in M9 minimal medium upon induction with arabinose (0,4% final concentration).

Fig. 2. Comparison of three different variants of sfGFP ORFs during 6h culture of E. coli DH5α in the richest medium – SB/PKB upon induction with L-arabinose (0h) (0,4% final concentration).

Fig. 3. Comparison of three different variants of sfGFP ORFs during 6h culture of E. coli DH5α in the rich medium – LB upon induction with L-arabinose (0h) (0,4% final concentration).

Fig. 4. Growth rate of E. coli DH5α transformed with mentioned biobricks during 6h culture in LB and SB/PKB media at 37°C (Fig.2. and Fig.3). Protein expression was induced at OD600= 0,4, with L-arabinose (0,4% final concentration).

Fig. 5. Comparison of three different variants of sfGFP ORFs during 6h culture of E.coli DH5α in M9 minimal medium upon induction with L-arabinose (0h) (0,4% final concentration). Protein expression was induced at OD600= 0,8.

The result suggest that in E. coli cells growing in rich media the introduction of rare codons to a sequence coding for a well soluble protein at a moderate level (like in pBAD systems) is not sufficient to observe any significant decrease in the rate of its translation. The translational rate of sfGFP from inversely optimized ORF is higher or equal to sfGFP biosynthesized from the most frequent codons. 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

Assembly Compatibility:

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