Difference between revisions of "Part:BBa K3059639:Design"
 
(2 intermediate revisions by the same user not shown)
Line 9: Line 9:
  
 
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 curli fibers and AG43 (this part contains two transcriptional units with pBlind. The first is pBlind-curli, and the second is pBlind-AG43). 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.
 
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 curli fibers and AG43 (this part contains two transcriptional units with pBlind. The first is pBlind-curli, and the second is pBlind-AG43). 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===
 
===Source===
Line 16: Line 15:
  
 
===References===
 
===References===
 +
 +
1. 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
 +
 +
2. Jin, X., & Riedel-Kruse, I. H. (2018). Biofilm lithography enables high-resolution cell
 +
patterning via optogenetic adhesin expression. PNAS 115, 3698-3703. doi: 10.1073/pnas.1720676115

Latest revision as of 03:29, 21 October 2019


pBlind Curli + AG43


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2730
    Illegal XhoI site found at 607
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
    Illegal NgoMIV site found at 177
    Illegal NgoMIV site found at 4999
    Illegal NgoMIV site found at 6223
    Illegal NgoMIV site found at 6733
    Illegal NgoMIV site found at 6754
    Illegal AgeI site found at 3027
    Illegal AgeI site found at 5749
    Illegal AgeI site found at 6493
    Illegal AgeI site found at 6907
    Illegal AgeI site found at 7149
  • 1000
    COMPATIBLE 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 curli fibers and AG43 (this part contains two transcriptional units with pBlind. The first is pBlind-curli, and the second is pBlind-AG43). 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

1. 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

2. Jin, X., & Riedel-Kruse, I. H. (2018). Biofilm lithography enables high-resolution cell patterning via optogenetic adhesin expression. PNAS 115, 3698-3703. doi: 10.1073/pnas.1720676115