Part:BBa_K3093100

PlacIq-LacI-Plac-cI-λPR dual inverter

The dual inverter system consists of PlacIq-LacI-Plac-cI-λPR.

The lacI gene is expressed from an unregulated promoter Placlq, and its product LacI represses the Plac promoter. Likewise, the Plac promoter controls the expression of the cI gene, and its product CI represses the PR promoter. The PR promoter controls the production of the red fluorescent protein (mRFP1), which represents the‘output’for the circuit, whereas the chemical inducer isopropyl β-D-thiogalactopyranoside (IPTG), which binds to LacI tetramers and renders them unable to repress Plac, provides an external control over the network and thus represents the‘input.’

Usage and Biology

The dual inverter consists of lactose operon, CⅠprotein and λPR012. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a brand new composite part.

The LacI repressor is constitutively expressed which inhibits transcription from the pLac promoter in the absence of IPTG, thus λPR012 could be active. While the expression of the CI repressor can be induced by externally added IPTG and inhibits the transcription of λPR012, hence realizing the reversal of the function.

Characterization

In order to characterize PlacIq-LacI-Plac-cI-λPR dual inverter, ECUST_iGEMer constructed a dual-plasmid: pIN1-pIN2.

Figure.0 Gene circuit of inverter system 

Before co-expressing this dual-plasmid, we verified the function of pIN1 and pIN2 respectively. pIN1 was composed of PlacIq-LacI-Plac-cI, using GFP as the reporter gene. Since CI and GFP were both under the control of Plac, the expression of GFP was disturbed by CI according to the measurement of GFP fluorescence. However, we performed SDS-PAGE to confirm that the pIN1 exactly worked.

Figure.1 SDS-PAGE results of pIN1 

The SDS-PAGE results showed that for pIN1, cI (26.2 kDa) and Lac I (38.9 kDa) was successfully expressed after induction, indicating that the construction of pIN1 was preliminary successful.

pIN2 was relatively simple, just consisting of λPR012 and using mRFP as the reporter. In the absence of CI protein, λPR012 was constitutively active to express mRFP. So we could clearly recognized the positive colony via observing the color while construction (the red colony might be the positive one). After colony PCR and sanger sequencing, we incubated the positive colony in 5mL M9 containing 0.1% Kan at 37℃ for 12 hr.

References

[1]Yokobayashi Y, Weiss R, Arnold FH. Directed evolution of a genetic circuit. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16587-91. Epub 2002 Nov 25. PubMed PMID: 12451174; PubMed Central PMCID: PMC139187.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1229
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 3156
  Illegal AgeI site found at 3268
  
• 1000
  COMPATIBLE WITH RFC[1000]