Part:BBa_K2036014

RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag

CIII acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor lambda CII, thereby helping the phage to lysogenize the host bacterium.
This circuit is built to characterize CIII's function comparing to control group: RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015)

Fig1: CIII characterization circuit-test group

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 1550
  

Protein&promoter

--CII and pRE

CII ( BBa_K2036000 ) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFP-LVAssrAtag as CK to see if CII efficiently activate pRE.

Fig2: According to the Flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.

Fig3: We also did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

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.

Fig4: 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 tandemly expressed CIII can efficiently protect CII from being degraded by Ftsh.

Preliminary experiments of LVAssrA-tag

In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Rgistery ( BBa_J04500 ) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.

Fig5: From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.