Team:XMU-China/Engineering
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This year XMU-China endeavored to tackle and had solved problems during our engineering process, generating expected results. Our SALVAGE project has three main sections: algae treatment system, mussel treatment system and light-induced suicide (kill switch) system (see our design of SALVAGE project).
After the several cycles of “brainstorm, design, human practices, design improvement” (see our Human Practicespage for details), we decided on several enzyme candidates. In total, 99 new BioBricks were designed for our project (see our Partspage).
Countless efforts are made to ensure our design can function to attain our goals. Among them, BBa_K3739000, BBa_K3739014 and BBa_K3739030 are typically worth mentioning. These three BioBricks perform well and function as expected. All the related data are recorded below. Hopefully, they will make some contribution to the iGEM community.
his-HutH (BBa_K3739000)
HutH, which can catalyze L-histidine into trans-urocanate, is found in the liver of vertebrates and in bacteria such as Escherichia coli, Salmonella and Pseudomonas. Because of the toxic effect of urocanate to P. globosa, we chose the histidine ammonia-lyase (HutH) from Pseudomonas putida to solve the algae problem. J23100-B0030 (BBa_K081005) was used to construct the expression circuit (BBa_K3739031), and the HutH protein was purified then used to test the enzyme activity.
1.1 Agarose Gel Electrophoresis
Fig. 1. DNA gel electrophoresis image of expression circuit for HutH (BBa_K3739031).
Result: When we were building this circuit, regular PCR was used to certify that the plasmid was correct. Target bands (1877 bp) can be observed at the position around 2000 bp (Fig. 1).
1.2 SDS-PAGE
Fig. 2. SDS-PAGE analysis of protein HutH in lysate of Vibrio natriegens and the eluant.
Result: hutH gene was expressed successfully. Compared with the negative control groups (NC), the target bands of HutH (55.5 kDa) in the eluant (FR) can be observed at around 50 kDa (Fig. 2).
1.3 Enzyme activity
Method: Enzyme activity of purified HutH was measured spectrophotometrically by monitoring the increase of absorbance at 277 nm, due to the formation of trans-urocanate. The concentration of trans-urocanate was calculated from the standard working curve determined in advance (OD277 was plotted against varying concentrations of trans-urocanate).
Result: We got the OD277-time curve of the product trans-urocanate at 28 °C. Then we calculated relevant enzyme activity and drew 1/Enzyme activity and 1/[L-histidine] curves (Lineweaver-Burk Plot), from which we can obtain relevant Km and kcat of purified HutH (Fig. 3).
Fig. 3. The relationship of 1/enzyme activity and 1/concentration of L-histidine.
TnaA-his (BBa_K3739014)
Mussels can form byssus, a network of biofilms that attach to surfaces. However, indole can prevent mussels from attaching by inhibiting the biofilm formation. TnaA was chosen for its function of converting tryptophan to indole. Similarly, J23100-B0030 (BBa_K081005) was used to construct the expression circuit (BBa_K3739044), and the TnaA protein was purified then used to test the enzyme activity.
2.1 Agarose Gel Electrophoresis
Fig. 4. DNA gel electrophoresis image of expression circuit of TnaA (BBa_K3739044).
Result: After transferring the correct plasmid into Vibrio natriegens, colony PCR was used to select the positive clones. Target bands (1739 bp) can be observed at the position around 1500 bp (Fig. 4).
2.2 SDS-PAGE
Fig. 5. SDS-PAGE analysis of protein TnaA in lysate of Vibrio natriegens and the eluant.
Result: tnaA gene was expressed successfully. Compared with the negative control groups (NC), the target bands of TnaA (53.5 kDa) in the eluant (FR) can be observed at around 50 kDa (Fig. 5).
2.3 Enzyme activity
Method: The amount of indole produced by TnaA from tryptophan in a certain time was subsequently measured by indole reagent, and the concentration of indole was calculated from the standard working curve determined in advance (OD570 was plotted against varying concentrations of indole).
Fig. 6. The relationship of 1/enzyme activity and 1/concentration of indole.
Result: We got the OD570-time curve of the product indole at 28 °C. Then we calculated relevant enzyme activity and drew 1/Enzyme activity and 1/[indole] curves (Lineweaver-Burk Plot), from which we can obtain relevant Km and kcat of purified TnaA (Fig. 6).
(kill switch)
BlrA-GFP (BBa_K3739030)
To facilitate biosafety and biocontainment, BlrA, originated from Bacillus subtilis, was chosen for its function of producing ROS (reactive oxygen species) upon blue-light irradiation, which can cause cell death of the bacteria. For this part, a GFP sequence was fused to the C-terminal of BlrA in order to direct view the expression level of the toxin gene, and blrA-gfp was cloned into to pET-28a(+) vector. To verify the cytotoxicity of BlrA, colony forming units (CFUs) were counted for indicating cell viability as time progressed after inducing.
3.1 Agarose Gel Electrophoresis
Fig. 7. The result of regular PCR (plasmid was added as the template for PCR).
Result: When we were building this circuit, regular PCR was used to certify the correct plasmids. Target bands (1542 bp) can be observed at the position around 1500 bp (Fig. 7).
3.2 Measurement for killing effects of BlrA
Method: During the cultivation, inducer IPTG was used to control the expression of blrA-gfp. After the addition of IPTG, blue-light was also supplied to activate BlrA at the same time. Samples were taken out by every 2 hours after inoculation, and IPTG was added at the fourth hour of cultivation. GFP fluorescence intensity and CFUs of every sample were measured to represent the expression level and killing effects of BlrA, respectively.
Fig. 8. The cytotoxicity of BlrA was verified. (A) Variation tendency of normalized fluorescence density (RFUGFP/OD600) of Vibrio natriegens harboring blrA-gfp_pET-28a(+) with or without adding IPTG. (B) Variation tendency of CFUs after Vibrio natriegens harboring blrA-gfp_pET-28a(+) were induced with and without IPTG. Gray areas represent dark state while the blue areas represent illumination state.
Result: blrA gene was expressed successfully under the inducement of IPTG (Fig. 8A). Meanwhile, CFUs of induced group decreased sharply after IPTG addition compared to the non-induced group, of which CFUs arose continuously (Fig. 8B). The difference of CFUs between these two groups has verified that BlrA can be used as a toxic protein for killing engineered bacteria.