Difference between revisions of "Part:BBa K3059638:Design"
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This circuit incorporates the pBlind-EL222 system, which is sensitive to blue light. Constitutively expressed EL222 can activate pBlind under blue light, resulting in increased expression of the downstream curli operon. | This circuit incorporates the pBlind-EL222 system, which is sensitive to blue light. Constitutively expressed EL222 can activate pBlind under blue light, resulting in increased expression of the downstream curli operon. | ||
To assemble this circuit, we utilized 3G DNA assembly, which required individual 3G-compatible DNA parts (BsaI cutsites and proper sticky ends) as well as transcriptional units flanked by the proper Universal Nucleotide Sequences (UNS). To construct the transcriptional unit containing the synthetic curli circuit, we assembled csgBAC and csgEFG (which are divergent in E. coli) together, linking them with an RBS before csgE. This assembly required the creation of new sticky end (sticky end alpha, see parts pages BBa_K3059626, BBa_K3059627, and BBa_K3059612) in addition to William and Mary 2018 iGEM's sticky ends A-E. | To assemble this circuit, we utilized 3G DNA assembly, which required individual 3G-compatible DNA parts (BsaI cutsites and proper sticky ends) as well as transcriptional units flanked by the proper Universal Nucleotide Sequences (UNS). To construct the transcriptional unit containing the synthetic curli circuit, we assembled csgBAC and csgEFG (which are divergent in E. coli) together, linking them with an RBS before csgE. This assembly required the creation of new sticky end (sticky end alpha, see parts pages BBa_K3059626, BBa_K3059627, and BBa_K3059612) in addition to William and Mary 2018 iGEM's sticky ends A-E. | ||
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===Source=== | ===Source=== | ||
Latest revision as of 03:24, 21 October 2019
pBlind Curli Operon, Blue-Light Inducible
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2730
Illegal NheI site found at 51
Illegal NheI site found at 74
Illegal PstI site found at 1829 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2730
Illegal XhoI site found at 607 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2730
Illegal PstI site found at 1829
Illegal NgoMIV site found at 177
Illegal AgeI site found at 3027 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This circuit incorporates the pBlind-EL222 system, which is sensitive to blue light. Constitutively expressed EL222 can activate pBlind under blue light, resulting in increased expression of the downstream curli operon. To assemble this circuit, we utilized 3G DNA assembly, which required individual 3G-compatible DNA parts (BsaI cutsites and proper sticky ends) as well as transcriptional units flanked by the proper Universal Nucleotide Sequences (UNS). To construct the transcriptional unit containing the synthetic curli circuit, we assembled csgBAC and csgEFG (which are divergent in E. coli) together, linking them with an RBS before csgE. This assembly required the creation of new sticky end (sticky end alpha, see parts pages BBa_K3059626, BBa_K3059627, and BBa_K3059612) in addition to William and Mary 2018 iGEM's sticky ends A-E.
Source
Our synthetic curli operon is composed of csgBAC, an RBS preceding csgE, and csgEFG. See BBa_K3059619, BBa_K305920, and BBa_K3059621, respectively for information the sources of these parts. Additional promoters, RBS, and terminators used in this circuit were chosen from the 3G DNA part library created by William and Mary 2018 iGEM.
References
Jayaraman, P., Devarajan, K., Chua, T. K., Zhang, H., Gunawan, E., & Poh, C. L. (2016). Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic acids research, 44(14), 6994–7005. doi:10.1093/nar/gkw548