Firstly, we built two plasmids, pHY300PLK-P43-CA1-T7(from iGEM parts library No. BBa_K2232014) and pHY300PLK-P43-CA2-T7 (CA2 is the codon optimization product of CA1) using Snapgene and sent the sequence to GenScript for synthesis. The plasmids were amplified by DH5α, then extracted, purified, and verified by sequencing.

After chemical transformation of plasmids, the transformed Bacillus subtilis WB600 strain were cultured in optimized LB and SMM medium, and obtained crude enzyme solution of carbonic anhydrase by centrifugation and ultrasonic disruption. We name the carbonic anhydrase expressed by BBa_K2232000 as CA1, while name the carbonic anhydrase expressed by BBa_K4202004 as CA2. Then we detected the molecular mass of carbonic anhydrase by SDS-PAGE and coomassie blue staining.

Result: SDS-PAGE displayed bands of 37kDa for CA monomer, which didn' t exist in the control group.

Fig 1-1 SDS-PAGE and Coomassie brilliant blue staining results of whole protein lysates of strain CA1, strain CA2 and blank WB600. Lane 1: Protein Ladder; Lane2: CA2 strain grown in LB medium, Lane3: CA2 strain grown in SMM medium, Lane4: CA1 strain grown in LB medium, Lane5: CA1 strain grown in SMM medium, Lane6: blank WB600 strain.

We added a 6X His tag to the N-terminus of CA on the vector, and the CA was purified by agarose-nickel column affinity chromatography. SDS-PAGE analysis was performed on the flowthrough, washing fluid and eluent during the purification of CA1 and CA2 by Ni-column.

Result: The eluate of CA1 and CA2 had two obvious bands at 37kDa and 74kDa, which were monomer CA and dimer CA, respectively. However, the Ni-NTA resin had much non-specific protein binding, we can add a small amount of imidazole to reduce non-specific protein binding.

Fig 1-2 A: SDS-PAGE analysis of CA1 protein purified by Ni-NTA resin. Lane1: flowthrough, Lane2-4: the 1-4 washing fluid, Lane5-9: the 1-5 eluate, Lane10: protein Maker B: SDS-PAGE analysis of CA2 protein purified by Ni-NTA resin. Lane1-5: the 1-5 eluent, Lane6-8: the 1, 2 and 4 washing fluid, Lane9: the flowthrough, Lane10: protein Maker.

To measure the activity of CA, we used the modified Wilbur-Anderson's method [1]. As we know, CA can catalyze CO₂ hydration and at the same time release H⁺ reducing the pH. According to that, we chose bromothymol blue, an acid-base indicator that appears yellow when pH≤6 and blue when pH>7.6. Therefore, the color development of bromothymol blue can indirectly reflect the change of pH from 8.0 to 6.0 by CA.

Result: After adding icy saturated CO₂ solution for 10min, the color of the tubes containing crude enzyme solution CA1 and CA2 began to change, indicating that the pH of the solution began to decrease. After 10min, the tubes containing the crude enzyme solution showed significant discoloration, and after 5 days, the tubes containing the CA1 crude enzyme solution turned completely yellow, implying that the pH had decreased from 8.0 to 6.0 due to the formation of H⁺ during CO₂ hydration.

The activity of CA1 and CA2 were verified in this experiment, but the enzyme activities were weak, possibly due to low enzyme expression or insufficient concentration of unpurified enzyme. In addition, the catalytic rate of CA2 crude enzyme solution was lower than that of CA1（without codon optimization）.

Fig 1-3 The activity of CA detected by the Wilbur-Anderson's method. From left to right, the four tubes were pH=6.0 Tris-HCl buffer with bromothymol blue indicator, reaction system with blank WB600 lysate, CA2 crude enzyme solution, CA1 crude enzyme solution. A: Initial reaction solution（0min); B: 10min after adding ice-saturated CO₂ solution; C: 5d after adding ice-saturated CO₂ solution.

The common α-CA is a secreted monomeric protein with esterase activity that can catalyze the catalyzes the formation of p-nitrophenol from p-nitrophenyl acetate and show the color(yellow) [2]. Although it was reported that the CA naturally produced by Bacillus Halodurans TSLV1 had no esterase activity, we still wanted to test whether the recombinant CA expressed in Bacillus subtilis WB600 had esterase activity. In this part, we used the Nanodrop and microplate reader to measure the color reaction rates of the CA crude enzyme solutions. We also measured the esterase activity of purified CA solution in order to avoid influence of the other proteins in the cell lysis solution.

Result: The system with crude enzyme solution which quickly changed from colorless to yellow had an absorption peak at about 400nm(Fig.1-4 A). Absorption-time curve at 405nm showed that CA1 and CA2 crude enzyme solutions had high esterase activity(Fig.1-4B). For purified CA solution,the results showed that purified CA1 and CA2 both still had strong esterase activity(Fig. 1-4 C), which was inconsistent with the literature we checked. This may be because the recombinant CA expressed in Bacillus subtilis WB600 has both monomer and dimer forms, and some monomer CA own esterase activity.

Fig 1-4 Detection of esterase activity of CA. A: absorbance of the system with crude enzyme solution at different wavelengths, B: esterase activity of CA1 and CA2 crude enzyme solution, x: Time (min), Y: A_405nm, Blank: no bacterial split solution, WB600: lysate of blank WB600 was added, CA1: the lysate of transformant-CA1, CA2:the lysate of transformant-CA2, C: esterase activity of purified CA1 and CA2 enzyme eluent, x: Time (min), Y: A_405nm, Blank: no enzyme solution, WB600: lysate of blank WB600, CA1: purified CA1 enzyme solution, CA2: purified CA2 enzyme solution.

To test the ability of engineered Bacillus subtilis WB600 with CA genes to precipitate CaCO₃, we cultured the engineered bacteria in 30ml of LB medium at 25^oC for 3 days and added 5 ml of 100mM CaCl₂ solution on the first and second days. We filtered the culture medium through a Whatman filter paper to separate the bacteria and CaCO₃. The bacteria and CaCO₃ were dried and weighed, respectively. We can calculate CaCO₃ precipitation capacity of engineered bacteria by the formula: CaCO₃ dry weight (mg)/cell dry weight (g) [3].

After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2 transformant became turbid. The precipitate was filtered and dried. According to the the formula: CaCO₃ production capacity = CaCO₃ dry weight (mg)/cell dry weight (g), we found that the the precipitation efficiency of CA1 was higher than that of CA2.

Fig 1-5 A: The cultures after induction. B: Filtered and dried sediment products. C: CaCO₃ productions by CA1 and CA2 for 3d. D:Picture of precipitation under scanning electron microscope.

We obtained the CDS and protein sequence of ACCBP and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-ACCBP. The codon of SP-ACCBP was optimized for Bacillus subtilis and synthesized by GenScript, and the plasmid PHY-300PLK-SP-ACCBP(PHY-ACCBP) was constructed.
Considering the large quantity of protein we needed for function verification, we constructed the expression plasmid PET-28a(+)-ACCBP for E.coli in order to obtain the purified protein of high concentration for later experiments. Besides, we also constructed the palsmid Pet28a(+)-Δ16 ACCBP to explore the influence of N-terminal amino acids to protein folding.

To confirm that the optimized sequence could be produced by E. coli, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 ^o C overnight. The next day, the overnight-cultured solution was transferred into 5 ml kanamycin resistant LB medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl₂ was added to induce the protein expression at 8^oC

At the end of induction, the bacterial solution was centrifuged at 12000 rpm at 4°C and the bacteria were collected. After washing the precipitation once with PBS, the precipitation were resuspended in a 1.5ml EP tube with 1mL PBS (appropriate amount of PMSF can be added).The bacteria were disrupted by ultrasonic disruption until the suspension was clear. The products are used for SDS-PAGE.

Result: Acorrding to the result of SDS-PAGE, we can find that the protein were both in the supernatant and precipitation. And the more protein are in the precipitation, indicating that under this condition majority of the protein are insoluble.

Fig 1-6 SDS-PAGE and Coomassie brilliant blue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .

The overnight-cultured bacterial fluid was inoculated into 500 ml Kanamycin resistant LB medium at the ratio of 1:100. The solution was cultured at 37 ^° C , 220rpm until OD₆₀₀ reached 0.6-0.8 and protein was induced at 8 ^oC with 1mM IPTG, 1% glycerol and 1mM CaCl2 for 48h. Then we collect the bacterium and resuspend in a special protein extract buffer (containing 10% glycerol, 500 mM PH 7.5 Tris-HCl buffer, and 0.01mM PMSF ). The next steps are same as 1.2.2 above .
Then we used His-tag Protein Purification Kit(Beyotime) to purify our protein (refered to protocol)
Notes: In order to get a better protein expression efficiency, we also use palsmids Pet28a(+)-ACCBP, Pet28a(+)-Δ16 ACCBP, strains BL21(DE3) and shuffle T7 for this experiment .

Result: The purified ACCBP and Δ16 ACCBP protein were obtained by multiple combination of plasmids and strains. The protein will be used in further experiments.

Fig 1-7 SDS-PAGE and Coomassie brilliant blue staining results of protein purification. A: The purification result of ACCBP; B: The purification result of Δ16 ACCBP. The results show that both proteins have a considerable abundance in eluent 2 and 3 although there are some non-objective bands shown due to proteolysis.

In order to verify the structure of active ACCBP protein, we conducted chemical crosslinking experiment. Add 0.1% and 0.5% glutaraldehyde to 25μl ACCBP protein eluent, mix and let it stand at room temperature. Take samples at regular intervals. After terminating the reaction with loading buffer, run 12% SDS-PAGE to detect the cross-linking structure.

Result: According to the result of SDS-PAGE, we found that that the band of 140kDa was the most significant one of all samples. As the apparent molecular weight of ACCBP monomer is about 25kDa,we predicted that ACCBP probably assembles as pentamers in the solution. And the cross-linking results were better with less non-objective bands when higher glutaraldehyde concentration was used.

Fig 1-8 SDS-PAGE and Coomassie brilliant blue staining results of ACCBP chemical crosslinking experiment.

Add an appropriate amount of ACCBP solution, water and MgCl₂ solution into the newly prepared saturated Ca(HCO₃)₂[4] to prepare 20μl solution containing 0, 8.0, 16.0, 40.0 mM MgCl₂. Drip the 20μl reaction system onto the cover glass. After 24 hours of reaction, observe the calcium carbonate crystals on the slides were studied by SEM.

Result: For better images, the calcium carbonate crystals were observed in different magnifications. It was obvious that when the concentration of Mg²⁺ was low, ACCBP induced the formation of typical square calcite crystals. When the concentration of Mg²⁺ reached 40mM, ACCBP induced the formation of more aragonite particles. Our results also indicated that the deletion of 16 N-terminal amino acids of ACCBP had no significant effect on the protein function.

Fig 1-9 The influence of Mg²⁺ to the crystallization results of ACCBP

The engineered SpyTag and Spycatcher protein sequences and CDS sequences were obtained from NCBI database. We designed two fusion proteins, HA Tag-GS-mRFP-GS-SpyTag and 6*HisTag-GS-Spycatcher-GS-mRFP, and commissioned GenScrip to synthesize the DNA sequences of the above two proteins. The sequences were inserted into Pet28a(+) to obtain new recombinant plasmids.

The above plasmids were transformed into BL21(DE3) strain respectively. After cultured in kanamycin-resistant LB plates at 37°C overnight, a single colony was picked and inoculated into kanamycin resistant LB medium for overnight culture. The next day, the overnight-cultured solution was transferred into 5ml kanamycin resistant LB medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6-0.8. Then 1 mM IPTG was added to induce protein expression under following conditions. And we will also set up a control group .Then we obtained crude protein solution by centrifugation and ultrasonic disruption.The products were used for SDS-PAGE .

Results: Acorrding to the result of SDS-PAGE, we found that the more protein were in the supernatant . Considering the concentration and quantity of the targeted protein ,we chose the condition of group 2 for the purification experiment.

Fig 2-1 SDS-PAGE and Coomassie brilliant blue staining results of supernatant and sediment of BL21(DE3) strain. A: Pet28a(+)-HA tag-GS-mRFP-GS-Spytag B: Pet28a(+)-His-tag-GS-spycatcher-GS-mRFP . The lanes and the target bands have been indicated.

The protein mRFP-SpyTag was induced at 20^oC for 10h, and then used HA-tag Protein IP Assay Kit with Magnetic Beads (Beyotime) to purify our protein (refered to protocol). The protein SpyCatcer-mRFP was induced at 20^oC for 10h, and then used His-tag Protein Purification Kit(Beyotime) to purify our protein ( refered to protocol). The products were used for SDS-PAGE analysis and later experiments.

Results:After repeated trials, we adjusted the composition of lysis buffer to obtain better purification efficiency. Finally, we successfully obtained the purified proteins. For mRFP-SpyTag, we found that the targeted band can be observed in Fig2-2 the lane 3, though the most of the targeted protein still reserve in the magnetic separation of supernatant after adsorption. For SpyCatcer-mRFP, we obtained a good purification effect according to the results of SDS-PAGE analysis. It was worth noting that in order to obtain better purification effect, we increased the content of imidazole in the eluation buffer (eluent(150 mM imidazole) ). The final results indicated that the protein with high purity was obtained in the eluent(150 mM imidazole).

Fig 2-2 SDS-PAGE and Coomassie brilliant blue staining results of HA-tag protein purification. Lane 1: Bacterial lysate(Input); Lane 2: Magnetic separation of supernatant after adsorption; Lane 3:Elution with loading buffer.

Fig 2-3 SDS-PAGE and Coomassie brilliant blue staining results of His-tag protein purification. The lanes and the target bands have been indicated.

The purified proteins were quantified by the BCA method (refered to protocol).Limited to laboratory equipment, we chose OD₅₇₈(the best wavelength is 562) for quantitative protein measurement. Then, we test the purified protein mRFP-spytag and spycatcher-mRFP, and get the concertration of protein by the standard curve roughly. It`s worthy to note that, we utilize the HA-peptide solution to elute the mRFP-spytag, so the result is not quite accurate.

Result:

Fig 2-4 The BCA-standard curve

the correlation between OD_578nm and the protein content(μg):

$OD578-μg: Y = 0.0446X R_2=0.98$

$OD578(\frac{1}{2} SpyCatcher-mRFP)=13.22$

$OD578(mRFP-SpyTag)=0.418$

$Protein concentration: $

$SpyCatcher-mRFP=1.3 mg/μl$

$mRFP-SpyTag=0.02 mg/μl$

The mRFP-spytag and spycatcher-mRFP protein samples were diluted to appropriate fold. We collocted 200μl of each, mixed and added 100μl 5×PBS. The mixture was incubated at 37 ^oC for 2h. Sample was collected for SDS-PAGE and WesternBlot analysis.It`s worthy to note that we choose exhibit the whole PDVF to abtain a visual outcome.

Result :In the lane of spy, we clearly found the signal of Spycatcher-SpyTag complex and corresponding protein. However, in the lane of Spycatcher-mRFP and mRFP-SpyTag, we only saw the signal of the protein possessing the corresponding tag. So proof the function of SpyTag-SpyCatcher.

Fig 2-5 The result of Western Blot by two kinds of antibody . Spy:The mixture of two protein after incubation spy

We obtained the CDS and protein sequence of EutM, engineered SpyCatcher and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-EutM-spycatcher. To ensure that the designed fusion protein still has the ability to assemble, I-TASSER was used for homology modeling. The results showed that the recombinant protein could still form a reasonable spatial structure.

Fig 2-6 The result of homologous modeling by I-TASSER

The codon of SP-EutM-spycatcher was optimized for Bacillus subtilis and synthesized by GenScript, and the plasmid PHY-300PLK-SP-EutM-spycatcher (PHY-EutM-spycatcher) was constructed. We ligated appropriate promoter and terminator sequence with CDS sequences by In-Fusion Clone strategy to construct plasmids, PHY-PsacB-SP-EutM-SpyCatcher-DT (DT means Double Terminator) and PHY-P43-SP-EutM-SpyCatcher-DT, respectively.

To screen the best conditions for the protein expression of B.subtilis, we employed SMM medium and LB medium for pre-expreiment. We transformed the plasmid PHY-P43-EutM-spycatcher-DT into Bacillus subtilis WB600 by chemical transformation (refered to protocol), inoculated in tetracycline-resistant LB plates and cultured it at 37^oC overnight, picked single colonies, and cultured them in tetracycline resistant LB or SMM medium at 37^oC overnight.

Result: As shown in the table, SMM medium maintained a relatively stable pH value, and the final concentration of the bacteria reached 5 times that of LB medium, which is thought to be an increase in pH to inhibit the growth of bacteria in LB. Subsequent experiments will be cultured with SMM medium.

Bacillus subtilis transferred with plasmids was cultured in SMM for 48h according to the above culture method. 14. Having harvested the cells, we analyzed the total protein in supernatant and pellet. However, a very low amount of secreted protein was observed in the supernatant compared to visible bands in the pellet, and we suspected that secreted and scaffolded EutM proteins were pelleting together with the cells.

After reviewing the literature, we conducted numerous tests to eventually discover the protein expression validation technique [5]. Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for SDS-PAGE analysis.The preparation of all four protein fractions is shown as a flow chart. The products obtained were used for SDS-PAGE analysis.

Fig 2-7 Experimental workflow used for the analysis of EutM scaffold building block secretion and expression by engineered Bacillus. subtilis .(i) culture supernatant, (ii) urea supernatant containing urea solubilized scaffolds that co-precipitated with cells, (iii) lysed cell pellets after solubilization of co-precipitated scaffolds, and (iv) total protein (secreted-precipitated and intracellular) from completely lysed cells with sonication followed by lysozyme treatment.

Besides, we utilized the purified mRFP-SpyTag to verify the function of SpyCatcher domain in EutM-SpyCatcher. The details of this experiment could be found in 2.1.5 .

Result: A significant change in the bacterial fluid properties was observed, which turned into a viscous liquid and the proteins attached to the bacteriophage could be pulled. It was thought to be a change brought about by EutM. According the outcome of SDS-PAGE , we clearly found the band of complex of EutM-SpyCatcher-mRFP-SpyTag(35 kD).

Fig 2-8 Observed EutM secretion property and SDS-PAGE results of EutM-SpyCatcher protein

Since the protein expressed by B.subtilis may be difficult to purify using Ni-NTA resin , due to protein folding, etc, we transformed PHY-P43-EutM-SpyCatcher-DT into E. coli strain in the hope of obtaining purified target protein for subsequent analysis. We transformed the above plasmid into BL21 (DE3) strain and cultured them in ampicillin-resistant LB medium at 37 ^° C for 24h. Product would be analysis by work flow in 1.2. His-tag Protein Purification Kit(Beyotime) was used for protein purification(refered to protocol), and the products would be used for SDS-PAGE and following experiments.

Results:

Fig 2-9 SDS-PAGE and Coomassie brilliant blue staining results of EutM-SpyCatcher protein expression in E.coil(left) and feature(right). Negative control was the result of empty E.coli .

Fig 2-10 His label recombinant protein purification effect. SU: supernatant lysate CL: cell lysate; FT: flow through; W1-3: wash 1-3; E1-5: elution 1-5

Concentrations of purified proteins were measured using the BCA Assay. For negative staining, 10 μL of protein was applied to the surface of a 200 μm formvar/carbon-coated copper grid . An equal volume of Trump’s fixative was added to the surface of the grid, and the protein/fixative drop was allowed to settle for 2 min. The surface of the grid was rinsed with 10 μL deionized water and excess fluid was removed. The protein on the grid was stained by applying 10 μL uranyl acetate (1%). Scaffolds were imaged on Phillips CM12 TEM with magnifications of x 20,000, x 50,000 and x 100,000.

Result: The TEM image shows that when we overexpressed and purified EutM, the isolated protein readily precipitated out of solution as large crystalline arrays, with obvious hexameric organization and symmetry. These results indicated that EutM could serve as a building block for the design of a protein-based scaffolding system.

Fig 2-11 Protein-based scaffolding system. Negative stain TEM of the white precipitate formed in the tube shows crystalline EutM scaffolds composed of hexameric tilesthat are assembled from structurally ordered arrays of EutM hexamers (red line indicates ordered crystalline lattice). Insert highlights individual EutM hexamers.

We obtained the CDS and protein sequence of Hag Bacillus subtilis from subtiwiki, and designed the recombinant protein Hag::SpyTag588. We employed overlapping PCR and In-Fusion Clone to ligated appropriate promoter and terminator sequence with CDS sequences to construct plasmids PHY-P43-Hag::SpyTag588-DT. The plasmids were transformed into BL21(DE3) strain. Following step the same as 2.1.3. The broken products were centrifuged, and the supernatant were collected and mixed with purified Spycather-mRFP, incubated at 37 ^°C for 2h. His-tag Protein Purification Kit (Beyotime) was used for protein purification and the products could be used for SDS-PAGE analysis.

Results:

Fig 2-12 SDS-PAGE and Coomassie brilliant blue staining results of His-tag protein purification. The target band of SpyCatcher-mRFP-Hag::SpyTag588 has been noted.

We tranformed the plasmid PHY-P43-Hag::SpyTag588-DT into Bacillus subtillis WB600 strain and inoculated into tetracycline resistant LB medium for overnight . Then we cultured the Bacillus subtillis WB600 containing PHY-Hag::SpyTag588 and PHY-EutM-SpyCatcher in the tetracycline resistance SMM medium. After culturing 36h, we added the 0.2 mg purified SpyCatcher-mRFP(The fluorescence probe can bind with Hag::spytag to charcterize the presence of the engineered Hag) per 5 ml medium into the medium and cultured the bacteria for 12h.Besides, we settled WB600 group and calcium carbonate group for control. Then we utilized the confocal laser scanning microscope (FLUOVIEW FV3000) to detect the cultures treated by SYTO .

Result: We can clearly observe the green fluorescence at 507 nm and red fluorescence at 610nm, while the group for control showed negative outcome. So we could conclude that the biological scafforld can be assembled reasonably.

Fig 2-14 Confocal laser scanning microscope of different groups. The cells were stained by SYTO for indicated.

Initially, we confirmed the function of immune part (NO.BBa_K302018) and the inhibitory concentration of subtilisin by zone of inhibition experiment and mixing subtilisin into the solid culture medium.

We first tested the inhibitory concentration of subtilisin by zone of inhibition experiment, using BL21 transformed by PHY-Immunity plasmid or PHY-Empty plasmid as control. The concentration of subtilisin were 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml.

Results: We could see from the radius of inhibition zone that the subtilisin needed quite a high concentration to work as an efficient antibiotic. Meanwhile, there was no obvious difference between the Immunity group and the control group.

Fig 3-1 Zone of inhibition experiment for subtilisin

Then, we prepared normal LB culture plates with different concentration of subtilisin, including 0.1mg/ml, 0.2mg/ml, 1mg/ml and 10mg/ml, in individual dishes. Then we inoculated BL21 transformed by immunity plasmid or PHY-empty plasmid as control in the plates.

Results:For the immunity group, some colonies grew on the surface of the media with 1mg/ml subtilisin, while the control group didn’t. This suggested that the PHY-Immunity plasmid worked well in BL21.

Fig 3-2The colonies growth condition in plates with different concentration of subtilisin

For signal module, we used GFP as the reporter gene to show the trigger of subtilisin by observing the bacterial colony’s phenotype and running SDS-PAGE.Therefore, we constructed PHY-Signal-GFP plasmid, using the biobrick BBa_K104001. Then we inoculated WB600 transformed by PHY-signaling-GFP plasmid on the media with or without 0.1mg/ml subtilisin.

Results:It’s quite clear that GFP was induced by 100μg/ml subtilisin, even though the growth of WB600 was moderately blocked by subtilisin.

Fig 3-3Phenotype of GFP-expressing colonies campared with the control group

In order to confirm the expression of GFP, we ran the SDS-PAGE later.

Results:Compared with the control group, the WB600 cultured with 0.1mg/ml subtilisin extended significantly strengthened expression of GFP.

Fig 3-4 SDS-PAGE to confirm the GFP expression

In general, although the promotor of signal module showed a little leakiness, it still worked pretty well in response to the subtilisin, especially in WB600.

Based on our design, a large population density led to low-oxygen environment, thus resulting in the expression of subtilisin production gene. Therefore, we compared the bacteria population and gene expression level between culturing in 15ml tubes and 50ml flask by measuring the OD600 and running SDS-PAGE.

Different from the previous part BBa_K302021, we built a new plasmid using the original Spa-box of strain ATCC6633, which was another Bacillus subtilis strain producing the subtilisin. Then we ligated the fragment with the linear vector containing Oxygen-limitation induced promotor and its RBS.

Fig 3-5Plasmid construction of Oxygen-limitation-SpaBCTS

Succeeding in constructing the plasmid(see new parts BBa_K4202007 ), we transformed it into our chassis for further protein expression. 50ml shaking flasks with 5ml culture media were introduced to reach an extra-large bacterial population while the normal 12ml shaking bacteria tubes were used as control.

Results:From the SDS-PAGE image, we could see a clear strip locating in 120kDa’s area for the 50ml shaking flasks group, revealing the excellent function of Oxygen-limitation induced promotor. However, it seemed that only the first gene (SpaB,120kDa) had expressed under the condition, suggesting that the original sequence didn’t match well to the promotor.

Fig 3-6Characterization of Oxygen-limitation induced promotor by SDS-PAGE

Unfortunately, due to the tight time schedule, we lacked time rebuilding the plasmid and producing the subtilisin.

In order to characterize the sucrose inducible activity of the sucrose inducible promoter PsacB, we extracted genomic DNA of Bacillus subtilis WB600, and amplified the PsacB sequence by PCR. The plasmid pHY300PLK-PsacB was constructed using In-Fusion clone. The mRFP:rrnB terminator:T7 terminator sequence coming from BBa_K143082 element was inserted into the 6bp position downstream of PsacB to obtain the pHY300PLK-PsacB-mRFP plasmid. At the same time, we also obtained the expression element containing Pveg-spoVG-RFP-rrnB T1 terminator-T7 terminator from BBa_K143082. The promoter Pveg is a constitutive promoter with medium activity. We also constructed it onto the pHY300PLK plasmid and obtained the pHY300PLK-Pveg-mRFP plasmid as a control of PsacB promoter strength.

Fig 4-1 A.the atlas of pHY300PLK-PsacB-mRFP plasmid. B.the atlas of pHY300PLK-Pveg-mRFP plasmid

Refering to the protocol provided by LMU, we transferred the pHY300PLK-PsacB-mRFP plasmid and the pHY300PLK-Pveg-mRFP plasmid into Bacillus subtilis WB600. Two positive clones were inoculated into 5ml tetracycline resistant LB medium and cultured overnight. The next day, the culture was transferred to 10mlLB medium at a ratio of 1:100. For both Pveg and PsacB promoter, characterization was carried out in LB medium containing 0%, 1%, 3%, 5% and 10% (w/v) sucrose.

The culture was incubated in shaking flask at 220rpm and 30 ^oC for 12h and centrifuged at 8000g, RT for 2min to harvest the cell. The expression of mRFP protein in harvested cells was first determined by naked eyes to roughly reflect the promoter strength.

Results:

It could been seen that the transcription initiation activity of Pveg promoter was much higher than that of PsacB promoter, and the PsacB promoter did not produce a visible mRFP accumulation.

Fig 4-2: A rough comparison of mRFP expression levels driven by different promoters under different induction conditions. A: the inducion result of Pveg promoter. B: the induction result of PsacB promoter.

The ultrasonic disruption product of the harvest cell was used to run the SDS-PAGE.

Results:It can be seen that the transcription initiation activity of Pveg promoter is much higer than PsacB promoter.The intensity of the mRFP band produced by PsacB promoter is very weak, and there is a certain degree of leakage expression when the induction activity do not exist under the condition of 5% sucrose induction.

Fig 4-3 the SDS-PAGE result of mRFP protein driven by different promoters under different induction conditions. CK: Bacillus subtilis with plasmid PsacB:EutM

To investigate the correlation between promoter activity and inducer concentration, we measured the bacterial fluorescence intensity at different inducer concentrations using sucrose and sucralose as inducers, respectively. The fluorescence producted by GFP were detected using flow cytometer.

Results: The flow cytometry results indicate that both sucrose and sucralose can positively regulate the activity of promoter. The activity of the promoter can increase up to ~5 times compared to the control when using sucrose as the inducer. However, due to the detection limit of flow cytometer, the data may not be very accurate. This may be due to the low activity of this promoter, too.

Fig 4-4 The fluorescence output after inducing using different types and concentrations of inducers. SUC: sucrose, SUL: suralose.

To enable higher promoter activity after induction, we also planned to engineered the PsacB promoter.

Through literature review, we found that the inducible activity of PsacB promoter comes from its ribonucleic antagonist sequence and the sacY protein that can bind to this sequence and stabilize its structure. [6] Based on this, we plan to iterate on the existing PsacB.

We introduce a strong constitutive promoter Pveg to replace the constitutive promoter of PsacB. To reduce the possible leakage of expression caused by the replacement of the strong promoter, we designed multiple repeat of the leader RNA sequence separated by spacers, and added spoVG RBS to enhance its expression. We constructed pHY300PLK-Pveg-N×LR vector containing 1× , 2× , 3× and 5× leader RNA sequence and used firefly luciferase or GFP as the reporter gene downstream of these promoters.

The characterization method is similar to the characterization method of PsacB. We also use flow cytomoter to measure the fluorescence produced by GFP.

Result:We characterized all our engineered promoters and found that the promoter activity decayed less under higher sucrose concentration conditions after replacing the constitutive promoter as well as multipling leader RNA (only in Pveg-2×LR promoter). Also, promoters with different numbers of leader RNA repeats showed different increases in activity after induction.The promoter Pveg-5×LR show the highest(more than 2 fold) induction activity.

Fig 4-5 The characterization result of our engineered promoters.

Our suicide switch consists of a sucrose-sensitive promoter and the mazE-mazF antitoxin-toxin system. We commissioned GenScript to synthesis the above sequence and construct it to the pHY300PLK plasmid.

Result:

Fig4-6 The structure of our kill switch

To characterized our kill switch, we transformed both the empty vector and the vector containing the kill switch into Bacillus subtilis WB600 and cultured them in the LB medium containing 1% sucrose. The OD_600nm(L=1mm) of the cultures was measured every 40 minutes when the OD_600nm(L=1mm) of cultures reached approximately 0.1.

Result:

Compared to the control group, the OD_600nm(L=1mm) of strains containing the kill switch began to decrease after the OD_600nm(L=1mm) reached 0.12. The early decrease of OD_600nm indicated that the bacteria are dying, which means our kill switch is working.

Fig 4-7 The growth curve of two Bacillus subtilis strains. Blue curve represents strains containing suicide switches and the yellow line represents strains containing the empty plasmid(CK).