Part:BBa_K3739066

J23106-B0034-K2332004-K2332002-B0030-blrA-GFP

light-regulated modules which can be induced to function or stop functioning by certain light. EL222 is the blue light dependent DNA-binding protein protein which controls pBLind. BlrA can be activated by blue light and produce reactive oxygen species(ROS).

Biology

EL222

EL222 is a photosensitive protein composed of an N-terminal light-oxygen-voltage (LOV) domain and a C-terminal helix-turn-helix (HTH) DNA-binding domain characteristic of LuxR-type DNA-binding proteins. In the dark, the LOV domain of EL222 represses its HTH domain. Illuminated by blue light (450nm), the LOV-HTH interaction is released, allowing EL222 to dimerize and bind to DNA.

pBLind

It is a modified luxI promoter. The lux box (LuxR and 3-oxo-C6-HSL complex binding region) of this promoter is replaced by the 18-bp EL222 binding region. Upon illumination with the blue light, EL222 dimer can presumably overlap the −35 region of the luxI promoter, thereby recruiting RNAP and activating transcription.

blrA-sfGFP

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.

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.

Usage and Design

This circuit and BBa_K3739117 were designed to test killing effect of switch with the light-induced promoter pBLind. By comparing the CFU(colony-forming unit) of induced and non-induced bacteria spead in plates, we managed to characterize the function of the blue light-regulated kill switch.

Characterization

1. Agarose Gel Electrophoresis

After constructing the part on the expression vector pSB1C3, the plasmid was transferred into E. coli BL21(DE3), then the positive colony were selected by chloramphenicol and verified by colony PCR and sequencing.

Meanwhile, we also constructed this part on the expression vector pET-28a(+), then transferred the plasmid into Vibrio natriegens. The constructed plasmid was transformed into Vibrio natriegens, the positive colony was selected by kanamycin and verified by colony PCR and sequencing.

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

2. CFU measurement

In order to simulate the possible leakage of engineering bacteria in actual condition and study the effectiveness of blue light strips in water outlet of cooling water circulation system in nuclear power plant, we designed a series of experiments to study the working state of the kill switch. The constructed genetic circuit was transformed into Vibrio natriegens through electroporation and was cultured in a dark environment throughout the whole process. After being cultured for 5.5 h in the shaker at 37 °C, samples were taken and gradient diluted to 10-7 for spread plate when its OD600 reached 1.0. After dilution, the samples were spread on four plates, two cultured in the dark and two illustrated by blue light.

After being cultivated for 12 h, we compared the growth situation of two groups (dark vs. illustrated by blue light). As shown in Fig. 2, many colonies were macroscopic in the group with the dark condition, while none visible colonies were observed in the group illustrated by blue light, indicating the success of the kill switch system.

Fig.2. Results of colonies culture under dark and illustrated by blue light. pBlind-EL222-pBlind-blrA-GFP (left), J23106-EL222-pBLind-GFP (right).

Fig.3. CFUs of V.natriegens carring J23106-EL222-pBLind-blrA-GFP and pBLind-EL222-pBLind-blrA-GFP. J+b:J23106-EL222-pBLind-blrA-GFP. p+b:pBLind-EL222-pBLind-blrA-GFP.

The process of engineering bacteria escaping from the working environment with a high concentration of bacteria to the natural environment with a low concentration of bacteria was simulated by lighting bacteria in the laboratory. Such an experimental scheme can verify the function of the combined circuit under the conditions close to the actual situation, that is, whether the engineered bacteria can be killed after being exposed to the blue light strips after leakage. According to the experimental results, the engineered bacteria can no longer produce progeny and form colonies illustrated by blue light, which proves that the kill switch system induced by blue light own an excellent performance.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1232
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 142
  Illegal AgeI site found at 367
  Illegal AgeI site found at 1084
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 983
  Illegal SapI site found at 1301
  Illegal SapI.rc site found at 1664