Composite

Part:BBa_K2036022

Designed by: Zhangyu Cheng   Group: iGEM16_HUST-China   (2016-09-20)
Revision as of 17:19, 19 October 2016 by ZhiZeng (Talk | contribs)


patp2-RBS-CI-TT-pR-RBS-CIII

This part is pH sensitive on account of pH sensitive promoter patp2. CI and pR are derived from bacteria lmabda operon. CI can block polymerase within pR so that transcription of CIII is inhibited. So it forms a NO gate of CIII expression. It serves as an intermediate to construct Prokaryote application circuit of Signal Filter([http://2016.igem.org/Team:HUST-China see HUST-China 2016 project]). This part is built by In-Fusion cloning method and together with BBa_K2036021 and BBa_K2036023 to further assemble BBa_K2036024

Fig1: Prokaryote application plasmid for lactose intolerance

Protein&protein reaction

We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.


Fig: FtsH degradation test of CIII

According to the Flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.


CI is a repressor from bacteriophage lambda. To test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFPLVAssrAtag-PET-Duet-1 and take pR-RBS-GFPLVAssrAtag-PET-Duet-1 as control to test its inhibition function. As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.

Fig: CI-pR inhibition test

We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below). From figure we can find the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.

Fig: Fluorescence detection


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
    COMPATIBLE WITH RFC[1000]


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