Signalling

Part:BBa_K3697008

Designed by: Lauren Ramlan   Group: iGEM20_Stanford   (2020-10-23)
Revision as of 20:18, 27 October 2020 by Cneimeth (Talk | contribs) (→‎Usage and Biology)


B. subtilis YFP CDS

This part is a yellow fluorescent protein optimized for use in Bacillus subtilis. It can be used as a reporter or signaling protein in B. subtilis or E. coli. Although it is optimized for expression in B. subtilis, E. coli are capable of translating this YFP. When highly expressed in B. subtilis, it will produce a signal that can be visualized using a plate reader. When highly expressed in E. coli, it produces a signal that can be seen with the naked eye. YFP has and excitation peak of 514 nm and emission peak of 527 nm.


Usage and Biology

YFP is a valuable tool for usage in B. subtilis due to its strong potential as a reporter protein. Colonies expression YFP at high levels from strong constitutive promotors such as pVeg can be quantified using a plate reader. This YFP can be expressed by both E. coli and B. subtilis, and is not tagged for degredation, giving a strong, enduring signal.

Due to the high levels of YFP expression from pVeg, this vector can be used to signal transformation, transcription, and translation in B. subtilis.

Stanford iGEM 2020 used this protein as a reporter for an RNA toehold based detection system. A plasmid expressing YFP by pVeg (BBa_K3697009) was linearized using PCR such that one end of the plasmid was pVeg and the other end was YFP. The insert, containing an the sequence encoding a toehold (BBa_K3697011), was flanked by 40bp of homology to pVeg, and 40bp homology to YFP. The linearized pVeg YFP backbone and the insert were then combined using Gibson assembly. The resulting product should be a toehold that regulates the translation of YFP, and is under pVeg expression. In the presence of the KanR gene or RNA, the toehold binds, enabling translation of the YFP. When enough target is present, YFP should be produced at levels visible using a fluorescent plate reader.

T--Stanford--YFP_and_mCherry_in_E_Coli.png Figure 1: E. Coli expressing YFP and mCherry (optimized for B. subtilis). Note: YFP is being expressed in the E. Coli on the left side of image. 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.rc site found at 644


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