Part:BBa_K2014005

AraC-pBAD->sfGFP_B

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 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 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_K2014005. B letters correspond to the most frequent codons in E. coli K-12 orfeome.

We have compared the translational efficiency of sfGFP_B ORF with its non-optimized ORF (Ba_K1481002- provided to iGEM in 2014 by Poznan_Bioinf team) and its inversely optimized form – sfGFP_W (BBaK2014006) 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 results 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, which indicates that E. coli translational apparatus can easily adjust and takes advantage from different tRNA pool. In contrast to rich media, in M9 minimal medium the codon optimization based on codon usage is important because E. coli cells cannot biosynthesize the appropriate tRNA molecules and keep up with translation from ORF composed of the least frequent codons with same efficiency as optimized ORF.

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