Difference between revisions of "Part:BBa K3145003:Design"

(→‎References from [https://parts.igem.org/Part:BBa_K2598027:Design]Bba_K2598027)
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===Design Notes===
 
===Design Notes===
−
There was an illegal XbaI cut site in between our T7 promoter and RBS region in our pY71-bFMO plasmid that was corrected through site-directed mutagenesis to make our construct identical to J18912-bFMO.
+
There was an illegal XbaI cut site in between our T7 promoter and RBS region in our pY71-bFMO plasmid that was removed through site-directed mutagenesis to make our T7 promoter and RBS region identical to J18912.
  
 
===Source===
 
===Source===
−
Our original construct (pY71-bFMO) was cloned in our lab. After we performed site-directed mutagenesis to correct a point mutation in our T7 promoter and RBS region, our original construct was now identical to the J18912-bFMO construct.
+
Our construct (pY71-bFMO) was cloned in our lab. The bFMO gene was obtained as a gBlock and cloned into the plasmid  pY71-sfGFP, to replace sfGFP. We performed site-directed mutagenesis to remove XbaI cut-site in our T7 promoter and RBS region.
  
 
===References===
 
===References===
 
*<p align="justify"> Kwon, Y.-C. & Jewett, M.C.High-throughput preparation methods of crude extract for robust cell-free protein synthesis. [https://www.nature.com/articles/srep08663|<i>Sci. Rep.</I>] 5,8663; DOI:10.1038/srep08663 (2015).</p>
 
*<p align="justify"> Kwon, Y.-C. & Jewett, M.C.High-throughput preparation methods of crude extract for robust cell-free protein synthesis. [https://www.nature.com/articles/srep08663|<i>Sci. Rep.</I>] 5,8663; DOI:10.1038/srep08663 (2015).</p>
−
===References from [https://parts.igem.org/Part:BBa_K2598027:Design|''Bba_K2598027'']===
+
===References from [https://parts.igem.org/Part:BBa_K2598027 "Bba_K2598027"]===
 
*<p align="justify"> Cho,H.J. et al. Structural and functional analysis of bacterial flavin-containing monooxygenase
 
*<p align="justify"> Cho,H.J. et al. Structural and functional analysis of bacterial flavin-containing monooxygenase
 
reveals its ping-pong-type reaction mechanism. J Struct Biol 175, 39-48,(2011).</p>
 
reveals its ping-pong-type reaction mechanism. J Struct Biol 175, 39-48,(2011).</p>
  
 
*<p align="justify">Fernandez-Rodriguez, J., Moser, F., Song, M. & Voigt, C. A. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology 13, 706-708 (2017).</p>
 
*<p align="justify">Fernandez-Rodriguez, J., Moser, F., Song, M. & Voigt, C. A. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology 13, 706-708 (2017).</p>

Latest revision as of 22:23, 21 October 2019

J18912-bFMO


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 240
    Illegal AgeI site found at 263
    Illegal AgeI site found at 567
    Illegal AgeI site found at 897
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 20


Design Notes

There was an illegal XbaI cut site in between our T7 promoter and RBS region in our pY71-bFMO plasmid that was removed through site-directed mutagenesis to make our T7 promoter and RBS region identical to J18912.

Source

Our construct (pY71-bFMO) was cloned in our lab. The bFMO gene was obtained as a gBlock and cloned into the plasmid pY71-sfGFP, to replace sfGFP. We performed site-directed mutagenesis to remove XbaI cut-site in our T7 promoter and RBS region.

References

  • Kwon, Y.-C. & Jewett, M.C.High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci. Rep. 5,8663; DOI:10.1038/srep08663 (2015).

References from "Bba_K2598027"

  • Cho,H.J. et al. Structural and functional analysis of bacterial flavin-containing monooxygenase

reveals its ping-pong-type reaction mechanism. J Struct Biol 175, 39-48,(2011).

  • Fernandez-Rodriguez, J., Moser, F., Song, M. & Voigt, C. A. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology 13, 706-708 (2017).