Difference between revisions of "Part:BBa K3145003:Design"
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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 | + | 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 | + | 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 | + | ===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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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
Fernandez-Rodriguez, J., Moser, F., Song, M. & Voigt, C. A. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology 13, 706-708 (2017).