Part:BBa_K2014008

AraC-pBAD->mRFP_W

Usage and Biology

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 and least frequent synonymous codon in all reading frames of E.coli K12 orfeome (BBa_K2014005, BBaK2014006), according to the codon usage table generated for us by Prof. W. Karłowski. We have found and show it in results section that in E. coli cells growing in rich media the introduction of very rare codons to the sequence coding for a well soluble protein like sfGFP at a moderate level (like in pBAD systems) is not sufficient to observe any significant decrease in the rate of its translation. We decided then to check if the same results will be observed in the case of a fluorescent protein of a different sequence. We designed two variants of mRFP ORFs. Optimized one - composed of exclusively the most frequent codons in E.coli orfeome (mRFP_B) and mRFP_W composed of synonymous rare codons (mRFP_W). In mRFP_W coding sequence appeared 9 AGA codons (arginine), 12 CTA codons (leucine), 9 ATA codons (isoleucine), 23 GGA codons (glycine), 12 TCA codons (serine) and 12 CCC codons (proline), which are codons that can reduce both the quantity and quality of the synthesized protein [Kane, 1995]. These rare codons represent almost 40% of all codons in mRFP_W coding 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 used as well in pBAD expression vectors (Invitrogen, Thermo-Fischer). This promoter is induced by L-arabinose.

Fig. 1. The scheme of two ORF versions of mRFP. The mRFP_B is composed of the most common codons in E.coli K-12 orfeome as the opposite mRFP_W is composed of the rarest ones.

We compared the translational efficiency of mRFP_B and mRFP_W ORF by measuring the fluorescence intensity of mRFPs encoded by different ORFs, which are under control of an identical promoter with an identical 5’UTR (Fig. 2).

Fig. 2. Comparison of different variants of mRFP ORFs after 18h of E.coli DH5α culturing in in two rich media, LB and SB-PKB and in M9 minimal medium upon induction with L-arabinose (0,4% final concentration). Protein expression was induced at OD600= 0,4.

These results vary from those obtained for sfGFP_B (BBaK2014005) and sfGFP_W (BBaK2014006) because we are able to observe significant decrease in the rate of translation from inversely transcribed ORF (mRFP_W comparing to optimized ORF (mRFP_B) in all three media (SB/PKB, LB and M9 minimal medium) after 18h of E.coli DH5α culturing. We suppose it might be the consequence of forming problematic secondary structures in mRNA transcribed from mRFP_W construct.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961