Part:BBa_K3739030

blrA-GFP

blrA can be activated by blue light and produce reactive oxygen species(ROS).

Biology

Flavin-combined fluorescent protein, coded by a kind of optogenetic toolbox LOV(light-oxygen voltage)-based photosensitizer gene, can induce the light-driven killing of bacteria. BlrA, originating from Bacillus subtilis, is a blue light-induced GTP-binding receptor, which possesses the LOV domain and autofluorescence. LOV domain enables it to be activated by blue light and produce ROS, which can damage the bacterial structure.

Gfp codes for a green fluorescence protein.The fluorescence density of GFP can reflect the expression level of BlrA.

Usage

To intuitionally show whether blrA expresses or not, it is linked to a GFP through a 3GS linker. The growth condition and the level of BlrA can be reflected by the intensity of green fluorescence.

Fig.1. The result of colony PCR. Plasmid pET28a(+).

We take blrA as the core of our kill switch, combine it with promotor, terminator and a series of necessary elements. The whole part is inserted into a plasmid backbone and finally transformed into E.coli to gain enough correct and functional circuits. The circuits are transformed into Vibrio.natriegens. We choose growth curve and CFU to measure the killing effect of BlrA.

Characterization

The blrA-gfp was cloned into pET-28a(+) for simplifying the inducing operations when verifying the cytotoxicity of BlrA.

In vivo assessment of BlrA-mediated phototoxicity in our engineered bacteria (V. natriegens) was implemented and colony forming units (CFUs) were counted for indicating the cell viability. In the experiment, a small part of bacteria culture was taken out to measure OD600 and GFP fluorescence intensity by every 2 hours after inoculation, and the samples were diluted then spread evenly over the surface of nutrient agar plates. When OD₆₀₀ reached ~0.8 (at 4th hour), IPTG was added and blue-light irradiation was turned on at the same time to induce the expression of BlrA and activate its phototoxicity, respectively.

Fig.2. The cytotoxicity of BlrA-GFP. (A) The RFUGFP/OD600 of different groups were calculated as time progressed. (B) The growth curves were measured as time progressed. (C) Calculated CFUs of the groups with/without IPTG added. (D) Calculated CFUs of the groups with IPTG added under different illumination circumstances. Gray areas represent dark state while the blue areas represent illumination state (if need). IPTG was added (if need) at the 4th hour after inoculation.

BlrA was expressed after inducing since the GFP fluorescence intensity (relative fluorescent unit, RFU) normalized to OD₆₀₀ (RFUGFP/OD₆₀₀) increased fast after 4 hours (Fig. 2A). And after IPTG inducement, the CFUs (counted at the time of 24 hours after plating) of the group exposed to blue-light showed a significant declined tendency while in contrast the CFUs of the group with no IPTG added increased as time progressed, which indicated that BlrA functioned as a toxin and resulted in cell death (Fig. 2C). However, the decrease of CFUs of the group with IPTG inducing while incubated in darkness was also observed (Fig. 2D). This implied that BlrA-GFP might have low or no light-response property, compared to simple BlrA.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 406
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 258
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 157
  Illegal SapI site found at 475
  Illegal SapI.rc site found at 838