Difference between revisions of "Part:BBa K2540015"
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===Usage and Biology===
 
===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.  
 
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.  
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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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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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Revision as of 21:31, 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.




Figure 11: 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).

The part was characterized in four E. coli strains, S. oneidensis , and P. putida 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.

Figure 1: Fluorescence levels of mKate2 under the control of the broad host range element (strength 7).


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.