Generator

Part:BBa_K2014012

Designed by: Daria Niewiadomska   Group: iGEM16_UAM_Poznan   (2016-10-12)

pxylF-xylA->sfGFP_W


Usage and Biology

We designed pxylF-xylA->sfGFP_W 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 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 a relatively weak xylose induced promoter- pxylF-xylA.


Fig. 1. The scheme of the biobrick BBa_K2014012. W letters correspond to the rarest codons in E. coli
K-12 orfeome.


We have compared the translational efficiency of sfGFP_W ORF with its non-optimized ORF (BBa_K1741007 - provided to iGEM by our team last year) and its optimized form – sfGFP_B (BBa_K2014011) 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_K2014011, BBa_K1741007, and BBa_K2014012 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).


Fig. 2. Comparison of three different variants of sfGFP ORFs during 6h culture of E. coli DH5α in rich media upon induction with D-xylose (0h) (0,4% final concentration).


Fig. 3.Photograph presenting pellets with supernatants after lysis (right) (See: [http://2016.igem.org/Team:UAM_Poznan/Experiments Methods]) after 5th hour of culturing E. coli cells in SB/PKB medium with 0,4% of D-xylose.


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


The results we obtained (Fig. 3, 4) suggest that in E. coli cells growing in both rich and minimal media, the introduction of rare codons into a sequence coding for a well soluble protein, expressed at a moderate level is not sufficient to observe any significant decrease in the rate of its translation. The translational rate of sfGFP from inverse optimized ORF is higher or equal to sfGFP biosynthesized from the most frequent codons. This indicates that E. coli translational apparatus can be easily adjusted or that such a gene takes advantage of this that it uses a different tRNA pool than highly expressed proteins do at the same time.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 437
    Illegal SpeI site found at 572
    Illegal SpeI site found at 749
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 437
    Illegal SpeI site found at 572
    Illegal SpeI site found at 749
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 153
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 437
    Illegal SpeI site found at 572
    Illegal SpeI site found at 749
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 437
    Illegal SpeI site found at 572
    Illegal SpeI site found at 749
  • 1000
    COMPATIBLE WITH RFC[1000]


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