Part:BBa_K2036018

pRE-RBS-Cro-RBS-CII-TT-ptrp2

This is a part of Prokaryote version of Signal Filter characterization([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K2036027).

Fig1:Prokaryote version of Signal Filter characterization circuit

Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.

Fig2:Prokaryote version of Signal Filter characterization expression plasmid

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.

Fig3: CII and pRE activation test

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

Fig4: Fluorescence detection

Preliminary experiments of ptrp2

ptrp2( BBa_K2036024 ) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.

Fig5:According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.

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]