Part:BBa_K2036015

RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag
CII acts as a necessary and sufficient cis-acting target for rapid proteolysis by initiating pRE. The circuit is built to test CII and pRE interaction together with RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag( BBa_K2036013)and pRE-RBS-GFP-LVAssrAtag( BBa_K2036011) . Considering Ftsh protease degrades it raidly in the host, RBS-CII-RBS-CII tandom expression circuit is constructed.

Fig1:CII and pRE interaction characterization circuits

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 1770
  

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-GFPLVAssrAtag 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 tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.