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<p>When construct containing mKate2 with oRBS was transformed into <i>E. coli</i> MG1655, no fluorescence over background was detected. However, when mKate2 plasmid was co-transformed with a plasmid containing orthogonal 16S rRNA, ~100 fold increase of fluorescence compared to negative control was observed (Figure 1). This demonstrates that only orthogonal ribosome is able to translate the mRNA of mKate2.</p>
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<b class = "caption" ;"scale2">Figure 11: characterization of orthogonal translation constructs in <i>E. coli </i>.</b>  
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<b class = "caption" ;"scale2">Figure 1: characterization of orthogonal translation constructs in <i>E. coli </i>.</b>  
 
IPTG-dependent mKate2 fluorescence is observed when plamids containing o16S rRNA controlled by Plac promoter and oRBS-mKate2 are co-transformed into <i>E. coli</i> (left). At 0.1 mM IPTG, ~100 fold fluorescence over negative control is observed, demonstrating that translation is orthogonal (right).  
 
IPTG-dependent mKate2 fluorescence is observed when plamids containing o16S rRNA controlled by Plac promoter and oRBS-mKate2 are co-transformed into <i>E. coli</i> (left). At 0.1 mM IPTG, ~100 fold fluorescence over negative control is observed, demonstrating that translation is orthogonal (right).  
 
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<p> The part was characterized in four <i>E. coli</i> strains, <i>S. oneidensis </i>, and <i>P. putida </i> using mKate2 fluorescent protein. Figure 1 shows fluorescence levels after cells transformed with a plasmid containing mKate2 under the control of the broad host range regulatory element were grown in LB for 24 hours.
 
  
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<p><b>Figure 1</b>: Fluorescence levels of mKate2 under the control of the broad host range element (strength 7).</p>
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Latest revision as of 21:35, 17 October 2018


Orthogonal RBS for expression across bacteria

This orthogonal RBS was designed using RBS calculator for orthogonal 16S rRNA predicted to function across a variety of bacterial strains. Prediction was done by algorithm written by Rice iGEM 2018 team based on previous work by Chubiz & Rao.


Usage and Biology

The orthogonal RBS contains an altered Shine-Dalgarno sequence, which prevents binding of the wild-type 16S rRNA subunit. Only 16S rRNA subunits containing a complementary orthogonal anti-Shine Dalgarno sequence may bind to the orthogonal RBS. This RBS along with its corresponding 16S rRNA may be used when orthogonal translation is desired in bacteria.


When construct containing mKate2 with oRBS was transformed into E. coli MG1655, no fluorescence over background was detected. However, when mKate2 plasmid was co-transformed with a plasmid containing orthogonal 16S rRNA, ~100 fold increase of fluorescence compared to negative control was observed (Figure 1). This demonstrates that only orthogonal ribosome is able to translate the mRNA of mKate2.



Figure 1: characterization of orthogonal translation constructs in E. coli . IPTG-dependent mKate2 fluorescence is observed when plamids containing o16S rRNA controlled by Plac promoter and oRBS-mKate2 are co-transformed into E. coli (left). At 0.1 mM IPTG, ~100 fold fluorescence over negative control is observed, demonstrating that translation is orthogonal (right).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal BsaI.rc site found at 58


References

Chubiz, L. M., & Rao, C. V. (2008). Computational design of orthogonal ribosomes. Nucleic Acids Research, 36(12), 4038–4046.