Part:BBa_K2387073

Inducible GFP quenched by REACH2

This construct is designed to express for a dark quencher for GFP (E0040) whose quenching process takes place via a FRET system and for a TEV protease able to cut the linker binding the GFP fluorophore to its REACH2 queching molecule. The YFP quenching molecule fused to GFP reduces the observed fluorescence of GFP. Both GFP and YFP are fused with the linker K1319016 which includes a specific TEV protease (K1319004) cleavage site. The fused proteins brings GFP and REACh2 in proximity to each other which allows both GFP and REACh2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is absorbed by REACh2 leading to a reduction in the GFP fluorescence. When the linker fusing both proteins is cut by the TEV protease, the GFP fluorescence can be recovered leading to a strong fluorescence signal after the linker is cut. Once both proteins are separated, the FRET interaction is prevented resulting this in a GFP fluorescence response.

Two bacterial cell populations (BCPs) were co-transformed with an inducible COLE7 lytic mechanism (BBa_K2387066) to allow cell lysis after the induction of L-arabinose. COLE7 lytic mechanism is expressed under the pBAD promoter which is an E.coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [2]. However, when arabinose is addedd to the medium, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [3]. pBAD promoter allows then for the control of the lysis process to take place. Both BCPs were combined in equal concentrations and a lytic process was induced which should allow the TEV protease to cut the linker fusing both GFP to its quenching molecule (REACh2 or the mutated M2 fragment). Once both proteins are separated, the quenching interaction is prevented resulting in an increase in GFP fluorescence.

The induction of lysis via the addition of L-arabinose lead to the breakage of the bacterial cell membranes (Figure 1). Fluorescence is increased in comparison to the non co-transformed strains which are not able to lyse, suggesting that the the lytic process leads to the recovery of the quenched GFP. This can indicate that both REACH2 and the mutated M2 fragment work as expected and that its quenching effect might be inhibited after the linker binding it to the GFP is cut, allowing GFP to recover its fluorescence (Figure 1).

Figure 2: Assessment of two proteolytic quenching mechanisms (GFP quenched by REACH2 or by a mutated version of M2 fragment). GFP-REACH2 stands for a strain expressing the GFP fluorophore quenched by REACH 2, GFP-M2 stands for a strain expressing the GFP fluorophore quenched by M2, TEV strain stands for a constitutively expressed TEV protease strain, TEV + GFP-REACH2 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains, TEV + GFP-M2 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains, GFP-REACH2 & COLE7 stands for a strain expressing the dark quenched GFP and co transformed with the COLE7 lytic mechanism, GFP-M2 & COLE7 stands for a strain expressing the dark quenched GFP and co transformed with the COLE7 lytic mechanism, TEV + GFP-REACH2 & COLE7 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains both of them co transformed with the COLE7 mechanism and TEV + GFP-M2 & COLE7 stands for a combination of TEV protease expressing strains and GFP quenched expressing strains both of them co transformed with the COLE7 mechanism.

References

[1] L. H. Burch, L. Zhang, F. G. Chao, H. Xu, and J. W. Drake, “The bacteriophage T4 rapid-lysis genes and their mutational proclivities.,” J. Bacteriol., vol. 193, no. 14, pp. 3537–45, Jul. 2011.

[2]

Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.

[3]

Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1342
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1282
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

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