Composite

Part:BBa_K4192130

Designed by: Jiahui Li   Group: iGEM22_CAU_China   (2022-09-30)
Revision as of 16:10, 9 October 2022 by Subconsciousness (Talk | contribs) (→‎Biology)


Plac-RBS-yf1-RBS-fixJ-Pfixk2-RBS-mcherry

The gene yf1, fixJ and PfixK2 make up the system of blue light regulation. Under dark conditions, phosphate groups are transferred from Yf1 protein to FixJ protein, and the phosphorylated FixJ protein activates the PfixK2 promoter, which in turn activates downstream gene expression. Under light induction, the phosphorylation of Fixj protein was blocked and the expression of genes regulated by the PfixK2 promoter was suppressed.The mCherry protein emits red fluorescence to characterize the efficiency of control elements in regulating gene expression.

Usage

Light control system can regulate gene expression by light intensity. MCherry has an excitation wavelength of 580nm and an emission wavelength of 610nm. Quantitative detection of mcherry can be achieved by detecting the optical signal at 610nm. This composite part was constructed to test the ability of the original light control system to regulate gene expression compared with the light control system containing yf1 mutation(BBa_K4192132).

Biology

In the absence of light, FixL first undergoes autophosphorylation at histidine at position 29, and then transfers the phosphate moiety to its homologous, noncovalently bound response regulator FixJ. Phosphorylated FixJ binds to homologous promoters to activate transcription; In the presence of light, FixJ cannot be phosphorylated, no longer binds to the promoter, and transcriptional activity is downregulated.[1]

Yf1-p.png

Fig.2 Regulatory mechanism of YF1 construction[1] Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 210
    Illegal SpeI site found at 1361
    Illegal SpeI site found at 2257
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 210
    Illegal SpeI site found at 1361
    Illegal SpeI site found at 2257
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 210
    Illegal SpeI site found at 1361
    Illegal SpeI site found at 2257
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 210
    Illegal SpeI site found at 1361
    Illegal SpeI site found at 2257
    Illegal NgoMIV site found at 764
    Illegal NgoMIV site found at 836
    Illegal NgoMIV site found at 926
    Illegal NgoMIV site found at 944
    Illegal NgoMIV site found at 1450
    Illegal NgoMIV site found at 1743
    Illegal NgoMIV site found at 1837
    Illegal AgeI site found at 478
    Illegal AgeI site found at 1618
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1507
    Illegal BsaI.rc site found at 377


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