Team:XMU-China/Proof Of Concept
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Overview:
In order to eliminate the threat from algae bloom to the nuclear power plants, we designed a series of the genetic circuits, including SP-his-CBM-hutH (BBa_K3739008 and BBa_K3739011), Aly01-his-3×SpyTag-lecA ((BBa_K3739005), and Aly01-his-3×SpyCatcher (BBa_K3739004). The function of SP-his-CBM-hutH is to inhibit the growth of single cells of P. globosa, while the function of Aly01-his-3×SpyTag-lecA and Aly01-his-3×SpyCatcher is to capture colonies and form hydrogel sediments.
Characterization in Vibrio natriegens:
According to the parts above, six kinds of composite parts were designed, including BBa_K3739031, BBa_K3739071, BBa_K3739106, BBa_K3739109, BBa_K3739114 and BBa_K3739115. BBa_K3739071 and BBa_K3739031 were transformed into Vibrio Natriegens to verifiy the function of SP-his-CBM-hutH. GE AKTA Prime Plus FPLC System was employed to purify the protein.
To compare the efficiency between His-HutH and his-CBM-HutH when killing algae, the purified protein of his-HutH and his-CBM-HutH was added to the shake flask containing 50 mL Phaeocystis globosa culture solution, separately. After being cultivated at 20℃ for 48 h, the team member extracted chlorophyll from algal cells with acetone. The absorbance of chlorophyll was measured at 665, 645, and 630 nm to calculate its concentration, which was used to indicate the distinction of inactivation ability between two proteins.
BBa_K3739114 and BBa_K3739115 were assembled into the plasmid backbone and transformed into E. coli BL21 (DE3), respectively. The two crude proteins were obtained through ultrasonication and mixed for incubation by bacterial fragmentation. The two crude proteins were mixed and incubated for some time. Western blot will be used to validate the binding between SpyTag and SpyCatcher in future experiments.
Based on functions validation of the toxic protein BlrA and blue light-induced system EL222 independently, blue light-induced circuits (pBlind-EL222-pBlind-blrA-GFP and J23106-EL222-pBlind-blrA-GFP) were further studied to verify the function of these two circuits. Positive colonies were selected by kanamycin preliminarily and then verified by regular PCR (Fig. 1) and sequencing.
Fig. 1. The result of regular PCR: BBa_K3739066_plasmid pET-28a(+) (left) and BBa_K3739117_plasmid pET-28a(+) (right). 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 or illustrated by blue light: pBlind-EL222-pBlind-blrA-GFP (left), J23106-EL222-pBLind-blrA-GFP (right). Fig. 3. Comparable colony count results of J+b (J23106-EL222-pBLind-blrA-GFP) and p+b (pBlind-EL222-pBlind-blrA-GFP) cultured under dark or illustrated by blue light. 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 after illustrated by blue light, which proves that the kill switch system induced by blue light own an excellent performance.
