Team:XMU-China/Results
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What work we have done:
We have verified that his-HutH enables the conversion L-histidine to trans-urocanate.
We have verified that his-CBM-GFP could bind to cellulose
We have verified that his-CBM-HutH could bind to cellulose and conversion L-histidine to trans-urocanate.
Work has not finished yet:
The function of sp-3SpyTag-lecA wasn't verified
The function of sp-3SpyCatcher wasn't verified
his-HutH
The hutH gene from Pseudomonas putida was heterologously expressed in chassis bacteria to produce histidine ammonia-lyase (HutH), which could catalyze L-histidine to trans-urocanate. Promoter (BBa_J23100), RBS (BBa_B0030), hutH gene, and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the HutH.
The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by regular PCR (Fig. 1) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the HutH from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2).
Fig. 1. The result of regular PCR. Plasmid pET-28a(+).
Fig. 2. SDS-PAGE analysis of protein in lysate of Vibrio natriegens and the eluant. Target bands (55.7 kDa) can be observed at the position around 50 kDa.
The absorbance of L-histidine was measured at 277 nm (ε = 18000 (mol⋅L-1)-1⋅cm-1) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and kcat (Fig. 3).
Thus, these data demonstrate that HutH works well to catalyze L-histidine to trans-urocanate.
Fig. 3.The relationship of 1/Enzyme activity and 1/concentration of L-histidine.
his-GFP-CBM
Proof of expression
Cellulose is a part of the structure on the surface of the Phaeocystis globose, and we realized that it could be a target for our protein 1. As described in the literature, as a part of the cellulolytic multi-enzyme protein, CBM owns the ability of cellulose-binding, which guides our functional proteins to bind around the single cell of Phaeocystis globose. We also call CBM the cellulose (or carbohydrate) binding module 2. So, the genetic circuit of his-GFP-CBM (K3739033) was designed, the expression product could bind to cellulose with fluorescence. Promoter (BBa_J23100), RBS (BBa_B0030), his-GFP-CBM gene and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-GFP-CBM.
The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (Fig. 4) and sequencing. Then, the colony with the corrected sequence was cultivated to express his-GFP-CBM. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the CBM from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 5).
Fig. 4. The result of colony PCR. Plasmid pET-28a (+).
Fig. 5. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 50.9 kDa.
Target bands can be seen at about 50.9 kDa, which verified the successfully expressed of His-GFP-CBM in Vibrio natriegens. The purified GFP-CBM and BSA were added to the quantitative filter paper (equality of mass), and the changes of protein concentration were measured after being incubated for a specific time. The data could indicate the activity of his-GFP-CBM. The experimental steps are as follows:
Firstly, quantitative filter paper fragments with an equal weight of 0.16 g were weighed and placed in the glass container, 2 mL GFP-CBM and BSA were added, respectively (Fig. 6). After being incubated on a shaker (20 min, 60 rpm) at room temperature, the changes of protein concentrations of GFP-CBM and BSA were measured using Bradford reagent (Fig. 7)
Fig. 6. Schematic diagram of experimental process.
Fig. 7. The concentration changes of BSA and his-CBM-GFP. ** = p < 0.01
his-CBM-HutH
In order to eliminate threat from Phaeocystis globose to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga. The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from Pseudomonas Putida were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of P. globosa and catalyzes L-histidine, secreted by P. globosa to trans-urocanate. which then elevates the ROS around the P. globosa to damage the algal cells.
Promoter (BBa_K525998), RBS (BBa_B0030), his-CBM-HutH gene (BBa_K3739071), and terminator (BBa_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (Fig. 8) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the CBM from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 9). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.
Fig. 8. The result of colony PCR. Plasmid pET-28a (+).
Fig. 9. SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.
The absorbance of trans-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the L-histidine solution, and the value OD277 was monitored and recorded. As shown in Fig. 10, the OD277 in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes L-histidine to trans-urocanate.
Fig. 10. Time course of the value of OD277.
What work we have done:
We have verified that tnaA-his enables the conversion of tryptophan to indole.
We have verified that polyphenol oxidase (PPO) can be expressed in E. coli and Vibrio natriegens.
We have verified that PPO could catalyze dopamine to dopaquinone.
We have verified the plastic binding ability of Vibrio natriegens with plastic-binding proteins on their surface.
Work has not finished yet:
The function of rhlAB wasn't verified.
The function of LCIKR2-GFP wasn't verified.
TnaA-his
As we know, mussels can form byssus, a biofilm that attaches to the solid surface. At the same time, indole can prevent mussels from attaching by inhibiting biofilm formation3. Thus, the tnaA-his brick (BBa_K3739044) was designed and constructed, which converts tryptophan to indole and thus inhibits the formation of mussel byssus.
Fig. 11. Gene circuit of tnaA-his.
The constructed plasmid was transformed into E. coli DH5α. Positive colonies were selected by chloramphenicol preliminarily and then verified by colony PCR (Fig. 12). The colony, confirmed by sequencing finally, was cultivated to amplification the plasmid. Then, the plasmid was transformed into the Vibrio natriegens by electroporation. The colony, verify by colony PCR (Fig. 13) and confirmed by sequencing finally, was cultivated to express the target protein.
Moreover, the total protein was gained from the supernatant after ultrasonication and centrifugation. GE AKTA Prime Plus FPLC System was used to harvest purified protein from the broken supernatant. An apparent protein peak in the AKTA FPLC System demonstrated the correct purified protein. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis and Coomassie blue staining (Fig. 14).
Fig. 12. Colony PCR of pET-28a(+)-J23100-RBS-tnaA-his in E. coli DH5α.
Fig. 13. Colony PCR of pET-28a(+)-J23100-RBS-tnaA-his in Vibrio natriegens.
As shown in SDS-PAGE of TnaA-his, the target protein (53.5 kDa) could be observed at the position around 50 kDa on the purified protein lanes and broken supernatant lanes, but not in the control groups. These results demonstrated that the tnaA with His-Tag was successfully expressed in Vibrio natriegens and purified using GE AKTA Prime Plus FPLC System.
Fig. 14. SDS-PAGE analysis of TnaA-his by Coomassie blue staining.
The purified tnaA enzyme activity was measure by the amount of indole produced from tryptophan within a specific time. The concentration of indole was calculated through the standard curve, which draws as the method as fellow:
After dissolving 5 mg of indole in a small amount of toluene, transfer the solution 50 mL volumetric flask, dilute with toluene to tick marks, and mix. Transfer 10, 20, 30, 40, and 50 µL solution above into five clean tubes respectively and dilute with toluene to 1.0 mL, then added 0.4 mL distilled water, 3mL PDAB (4-Dimethylaminobenzaldehyde) (5%, v/v) and n-butanol (5%, v/v) sulfate mixed chromogenic solution, and mix. Stand for 30 min and measure the absorbance at 570 nm. Taking the absorbance as the X-axis, the concentration as the Y-axis, draw the standard curve and obtain the equation.
20 µL PLP solution (0.20 mg/mL), 10 µL GSH solution (0.005 mol/L), and 270 µL tnaA enzyme solution were added in several 15 mL tubes respectively and then covered with 1.0 mL toluene. After keeping the temperature at 37 °C for 5 min, 100 µL tryptophan solution with different concentrations was added into the 15 tubes respectively, which were put in a shaker for 10 min at 37 °C. Then 3 mL of PDAB (5%, v/v) and n-butanol sulfate mixed chromogenic solution (5%, v/v) was added to stop the reaction. After 30 min, absorbance was measured at 570 nm, which was used to calculate the Km and kcat. (Fig. 14)
Fig. 15. The relationship of 1/enzyme activity and 1/concentration of Indole.
PPO-his
Proof of expression:
The mussel can adhere to a solid surface mainly through 3,4-dihydroxyphenylalanine (DOPA), the side chain in mucoprotein. We designed a genetic circuit to express polyphenol oxidase (PPO), which could degrade the DOPA. According to the literature, DOPA could be catalyzed by PPO to generate dopaquinone and significantly weaken mucoprotein viscosity. Thus, we express the PPO in Vibrio natriegens and characterization its enzyme activity, which is derived from Bacillus Megaterium (PDB ID: 3NM8). This PPO does not require a caddie protein for activity and possesses superior catalytic activity in the mixture of water and organic reagents. The brick of BBa_K3739015 has assembled into pET-28a(+) plasmid backbone with T7 promoter to express the PPO-his. After being verified by SDS-PAGE (Fig. 16), PPO was successfully expressed in E. coli BL21(DE3) and Vibrio natriegens.
Fig. 16. The result of regular PCR. Plasmid pET-28a(+).
Quantitative analysis of dopaquinone
The constructed plasmid was transformed into Vibrio natriegens through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR and sequencing. Then, the colony with the corrected sequence was cultivated to express PPO. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the PPO from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 17).
Fig. 17. SDS-PAGE analysis of PPO. Target bands (34.9kD kDa) can be observed at the position around 50 kDa.
The time course of the absorbance of dopaquinone was measured at 475 nm to obtain the data to calculate the enzyme activity.
Test system: sodium acetate buffer (170 µL), DOPA solution (20 µL, 50 mM) and PPO solution (10 µL) were added to the 96-well plate to measure the absorbance during the catalytic process through ECAN ® Infinite M200 Pro instrument. Parallel experiments were performed three times.
Kinetics of enzyme catalysis
The effects from different concentration of DOPA (0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM and 0.15 mM) on the PPO catalytic activity were investigated with 0.1 M sodium acetate buffer (pH=6.0). And the catalytic kinetic parameters of PPO were also obtained through calculation.
Fig. 18. The relationship of 1/Enzyme activity and 1/concentration of DOPA.
lpp-OmpA-LCI KR2-GFP
We attempt to display the plastic-binding protein on the surface of Vibrio natriegens through OmpA, which could endow the ability of plastic-binding to Vibrio natriegens. The gene of OmpA-LCI KR-2-GFP (Fig. 19A, BBa_K3739025) was assembled into the plasmid backbone and transformed into Vibrio natriegens through electroporation. After being verified by agarose gel electrophoresis, the plasmids have been transformed into Vibrio natriegens successfully (Fig. 19B).
Fig. 19. Gene circuit and agarose gel electrophoresis. (A) Gene circuit of OmpA-LCI KR-2-GFP (BBa_K3739025). (B) Target bands of OmpA-LCI KR-2-GFP (black arrow, 2100 bp).
After successful construction and transformation, the experiment was carried out to verify whether the OmpA-LCI KR-2-GFP was expressed and located on the membrane of Vibrio natriegens. Thus, the total membrane proteins were extracted to verify the expression of the OmpA-LCI KR-2-GFP. However, the complicated component of the membrane proteins also seriously hinders the analysis of SDS-PAGE (Fig. 20). The western blot analysis was performed, in which the GFP in fusion protein serves as the antigen, and the experimental results are shown in Figure 21. The targeted band was observed in the precalculated position in the western blot results, suggesting that the plastic-binding protein (OmpA-LCI KR-2-GFP) was successfully anchored on the surface of Vibrio natriegens.
Fig. 20. SDS-PAGE analysis of LCI KR-2-GFP by coomassie blue staining. Target bands of OmpA-LCI KR-2-GFP (red box, 61.7 kDa).
Fig. 21. Western Blot analysis of OmpA-LCI KR-2-GFP. Target bands of OmpA-LCI KR-2-GFP (white arrow, 61.7 kDa).
Fig. 22. Binding ability of engineered Vibrio natriegens to plastic. (A) The effect from adding plastic microsphere on the OD600 in experiment group (OmpA-LCI KR-2-GFP display on the surface of Vibrio natriegens) and control group (OmpA-GFP display on the surface of Vibrio natriegens). (B) Time course of ΔOD600.ΔOD600: The value of OD600 in each time minus the value of OD600 at the time of 0. **** = p < 0.0001.
Since some proteins in our project are intended to function in extracellular environment, how to get out of the cells is vital for realization of their functions. Signal peptide is a short amino acid sequence on secreted protein, which is a natural helper of protein to get out of cytoplasm. However, signal peptides can lead protein go to not only the extracellular environment but also the periplasm and membrane. There are several signal peptides selected for V. natriegens, but these are all directed into periplasm. 4 Although there is a signal peptide of alginate lyase from V. natriegens able to direct heterogenous protein to extracellular environment in E. coli chasis, It is not clear that there is a signal peptide conducting protein to extracellular environment in V. natriegens. 5
Selected procedure
Here, we use a tip to select the signal peptide. Download the translated CDS information of V. natriegens from NCBI Assembly. Through analysis of SignalP 5.0, we found out several candidate signal peptides which have significant likelihood to work with corresponding secretion system. According to the function of proteins containing proposed signal peptide, we can exclude the signal peptides targeting to the periplasm and cell membrane. Finally, we find out the best candidate signal peptide, LMT signal peptide, from our chassis.
Qualification results
To clarify whether the signal peptides we selected are able to direct the fused proteins to extracellular environment, we constructed Aly01 and LMT single peptides with BBa_K2560063 which is from 2018 Marburg iGEM program and transformed the pET-28a(+) plasmid with composite parts BBa_K3739038 and BBa_K3739041 into V. natriegens. Then we use the anti-GFP antibody to detect secreted GFP in the filtered supernatant of V. natriegens liquid culture by western blotting after V. natriegens is cultivated for .16 hours. The concentration of his-GFP in the supernatant of BBa_K3739041 is significantly higher than that in J23100-B0030-GFP. (Fig. 23A, 23B) Furthermore, less expression of BBa_K3739041 than that of GFP conforms that LMT-his-GFP is secreted rather than from lysed cells. (Fig. 23C) However, the concentration of Aly01-his-GFP is less than that of GFP. (Fig. 23A, 23B) It is probably because that there are less expression of Aly01-his-GFP in V. natriegens. (Fig. 23C) Thus, we use another method to prove our hypothesis.
Fig. 23. (A) Western Blotting detection of the V. natriegens supernatants of J23100-B0030-GFP, BBa_K3739038 and BBa_K3739041 after 16 h cultivation in LBv2 liquid culture, 37 ℃. Sup. means supernatant. (B) Grayscale analysis of (A). (C) Fluorescence intensity/OD600value of V. natriegens strain of J23100-B0030-GFP, BBa_K3739038 and BBa_K3739041 in LBv2 liquid culture after 16 h cultivation in 37 ℃.
Quantitation results
In order to exclude the difference of expression among J23100-B0030-GFP, BBa_K3739038 and BBa_K3739041 and quantify secretion effect of signal peptide, we treated V. natriegens liquid cultures after 12.5 h cultivation in 37 ℃ with 12.5 µg/mL chloramphenicol. Then we sampled the cultures every 6 minutes and terminated secretion with 20 mM NaN3. As time goes, the his-GFP concentration of culture supernatant were increased in both BBa_K3739038 and BBa_K3739041. This is indicated that both LMT and Aly01 signal peptide are able to translocate fused protein to extracellular environment. (Fig. 24A, 24B) We made a grayscale analysis for the modelling of secretion, and the total GFP is detected for confirmation that the translation is completely repressed. (See our Model pages)
Fig. 24. (A) Western blotting detection of sampled supernatants treated with chloramphenicol in different times. (B) GFP concentration of treated supernatant of BBa_K3739038 and BBa_K3739041 in (A)
Secretion in E. coli
Besides conducting protein secretion in V. natriegens, Aly01 signal peptide is reported that it is able to conduct protein secretion in E. coli as well. 5 We supposed that LMT signal peptide may also have conducting function in E. coli Thus, we used filtered culture of BL21(DE3) strain transformed with pET28a(+)-BBa_K3739043 and pET-28a(+)-BBa_K3739041 for SDS-PAGE. Then the SDS-PAGE gel is stained by silver staining. Contrasting to BBa_K3739043, his-GFP is detected in the supernatant of pET-28a(+)-BBa_K3739041.(Fig. 25) It is indicated that LMT signal peptide can also work for conducting secretion in E. coli.
Fig. 25. Silver staining result of supernatant of BL21(DE3) transformed with BBa_K3739043 and BBa_K3739041, respectively.
Then we used an alternative way to verify the secretion function of signal peptides in E. coli. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) could be detected with the biarsenical compound FlAsH-EDT2. 6
To quantify and qualify the secretion effect, FT was added into the C-terminal of BBa_K3739000, BBa_K3739006 and BBa_K3739009. Composite parts BBa_K3739084, BBa_K3739085, BBa_K3739087 were constructed (Fig. 26) on plasmid backbone pSB1C3 or pET-28a(+) and then the recombinant plasmid was transformed into E. coli BL21(DE3). Positive colonies selected by colony PCR and sequencing were cultivated for secretion analysis.
Fig. 26. Colony PCR results of BBa_K3739084, BBa_K3739085, BBa_K3739087
After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1 mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 /micro;M FlAsH-EDT 2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD 600. The result shows that the fluorescence intensity/OD600 of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well. (Fig. 27)
Fig. 27. Fluorescence intensity/OD600 of cell supernatant after incubation in the dark for 1h.
Moreover, to promote our understanding on secretion process mediated by signal peptides, we built a model about relationship between the amount of secreted recombinant protein and time. (See our Model pages) In order to get the value of PT (the total amount of recombinant protein per unit volume of reactor), P_M PM (the amount of the recombinant protein in the medium compartment in unit volume) and γ(extracellular secretion rate constant), a sets of characterization experiments were done.
BBa_K3739087 were constructed on plasmid backbone pET-28a(+) and then the recombinant plasmid was transformed into E. coli BL21(DE3). After cultivation for 5 hours and induction for 3 hours with 0.1 mM IPTG, 34 mg/L chloroamphenicol was added to bog down the synthesis of protein in the engineered bacteria in order to make the experimental operations more convenient. Then, the cell supernatant was collected in 0, 6, 12, 18, 24, 30, 40 min to test the amount of secreted recombinant protein by fluorescence detection of FlAsH-EDT2 and FT. (Fig. 28)
Fig. 28. The fitting of equations and experiments results.
Our kill switch system is mainly composed of an inducible promoter system (EL222-pBLind system) and a gene encoding toxin (blrA), both of which are blue-light activated for normally functioning (see our Design page). The toxin gene blrA is under the control of promoter pBLind that can be activated by EL222 upon blue-light illumination7. Besides, when exposed to blue-light, the toxin BlrA will be triggered to cause ROS (reactive oxygen species) formation then kill the engineered bacteria8.
Therefore, the engineered bacteria will survive and function as we designed when in the cooling water system of the nuclear power plant, where the environment is relative dark and the kill switch is in OFF state. Once the engineered bacteria escape from working environment and enter the regions with LED strips set in advance (see our Proposed Implementation page), the blue-light irradiation will turn on the kill switch system for achieving biosafety and biocontainment of our SALVAGE project.
What work we have done
The cytotoxicity of BlrA was verified.
The function of EL222 system that can be induced by blue-light was verified.
Cytotoxicity of BlrA
For direct viewing the real-time expression level of BlrA, a reporter GFP was fused to the C-terminal of BlrA via 3×GS linker for achieving co-expression. Thus, we could know the approximate expression level of BlrA of different sampling time-points by measuring GFP fluorescence intensity. 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. 29. 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. 1A). 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. 1C). However, the decrease of CFUs of the group with IPTG inducing while incubated in darkness was also observed (Fig. 1D). This implied that BlrA-GFP might have low or no light-response property, compared to simple BlrA.
Discussion for BlrA
BlrA, namely YtvA, is an FMN-binding fluorescence protein from Bacillus subtilis, which contains the photosensitive light-oxygen-voltage (LOV) domain and can cause lethal ROS formation in cells under the circumstance of rich-oxygen 9. However, our results showed above did not support this. Based on the phenomena we observed, we attributed this difference to the structural change of BlrA due to the fusion of GFP in the C-terminal of BlrA. Generally, conformational change and exposure of LOV domain might occur for BlrA during blue-light illumination, which results in ROS formation in a photosensitive manner. While fused with GFP, the LOV domain of BlrA might exposes consistently since the BlrA’s folding is influenced. Therefore, BlrA-GFP always shows cytotoxicity, whether being exposed to blue-light irradiation or not.
Additionally, the growth (OD600) of bacteria harboring blrA-gfp seemed not affected by the cytotoxicity after toxin gene was induced to express (Fig. 1B). However, this result supported that the killing mechanism of BlrA was not a cell-lysis manner but others. Due to potential time-lag of bacterial lysis and time-need for BlrA’s functioning, the OD600 could maintain in a relative stable level or increase slowly, albeit that cell death might has happened.
In conclusion, we showed that BlrA-GFP had cytotoxicity and could be used in the kill switch system.
The performance of EL222-pBLind system can be induced by blue-light
In order to obtain a photo-response system induced by blue-light, two genetic circuits of J23106-EL222-pBLind-sfGFP and pBLind-EL222-pBLind-sfGFP were constructed to verified the performance of EL222-pBLind in Vibrio natriegens. As a reporter protein, fluorescence intensity from GFP can represent the expression intensity of downstream gene in the two circuits with the condition of blue-light illumination and dark conditions respectively. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (BBa_K3739064), regular colony PCR (BBa_K3739065) (Fig. 30) and sequencing.
Fig. 30. The result of regular and colony PCR. Plasmid pET-28a(+). BBa_K3739064 (left) and BBa_K3739065 (right).
In the experiment, the two bacteria with different circuits were respectively set up in the uninduced (light avoidance) group and induced (blue light exposure) group. Samples were taken every two hours after inoculation, OD600 and fluorescence intensity were measured and recorded for 8 times. At the same time, the induced group of the two circuits illuminated by blue-light irradiation from time of 4.5 h after inoculation.
Fig. 31. EL222-pBLind system can be induced by blue-light. (A) The normalized fluorescence intensity (RFUsfGFP/OD 600) was measured as time progressed. J: J23106-EL222-pBLind-GFP, P: pBLind-EL222-pBLind-GFP, I: illumination, D: darkness. (B) The dynamic range of the two systems was compared by the RFUsfGFP/OD 600 at the last time-point of measurement. Gray areas represent dark state while the blue areas represent illumination state.
The RFUsfGFP/OD 600 value of the induced groups increased more than that of the uninduced groups over time after exposed to blue-light, indicating that the EL222 system harbors blue-light inducement property (Fig. 31A). By comparing the two groups of circuit J23106-EL222-pBLind-sfGFP alone, it was found that the leakage of circuit J23106-EL222-pBLind-sfGFP was already high without blue-light inducement, and the expression of sfGFP was not significantly increased after blue-light irradiation (Fig. 31A, pink lines).
At the same time, for the circuit pBLind-EL222-pBLind-sfGFP, since EL222 was almost not expressed in the uninduced condition, the sfGFP leakage of the circuit was relative low in the dark condition. In the induced group, a small amount of EL222 dimerized and bound to the promoter pBLind, greatly increasing the expression level of EL222, and then forming a positive feedback to enhance the expression level of downstream sfGFP, resulting in a rapid increase of RFUsfGFP/OD 600 value (Fig. 31A, blue lines). The higher dynamic range of circuit pBLind-EL222-pBLind-sfGFP was obtained, compared to J23106-EL222-pBLind-sfGFP (Fig. 31B). Similar phenomenon was observed when the two circuits were implemented in E. coli (see our Improve page).
In conclusion, the blue-light induced system based on transcription factor EL222 and its regulated promoter pBLind has excellent blue-light photo-sensitivity, which can keep the expression level of downstream genes in a relative low level in dark conditions. Once activated by blue-light illumination, a large number of downstream genes can be expressed via positive feedback regulation mechanism. Therefore, we hypothesized that the blue light-induced priming subsystem of pBLind-EL222-pBLind-effector protein was more suitable for the kill switch system (see our Proof of Concept page).
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