Week 1 (7.8-7.12)
1. Resuscitation and subculturing of bacterial gene donors:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture Bacillus subtilis BS-1, Agrobacterium tumefaciens C58, Microbacterium testaceum StLB037, and Solibacillus silvestris StLB214 from the stock culture in the LB media;
3) Culture at 30 ℃, 200 rpm.
Notes & Results:
Succeeded. Media became cloudy after 24 hours.
2. PCR amplification of the AHL lactonases from gene donors:
1) 50 μL PCR:
20 μL deionized water,
25 μL Phusion High-Fidelity PCR Master Mix (Thermo Fisher, Waltham, MA, USA),
2 μL forward primer (10 μM),
2 μL reverse primer (10 μM),
and 1 μL culture of S. pasteurii DSM33 as the template;
2) The PCR program:
a) one cycle of 98°C for 10 min;
b) 30 cycles of 98°C for 10s, 55°C for 30s, and 72°C for 3 min;
c) one cycle of 72°C for 5 min;
3) Run 1% gel electrophoresis to verify the size of amplified fragments;
4) Extract DNA from the gel using the TIANgel Midi Purification Kit (Tiangen, Beijing, China).
Notes & Results:
Succeeded. The first step of PCR (initial denaturation) was elongated to 10 min to ensure cell lysis and the release of genomic DNA template.
Week 2 (7.15-7.19)
3. Recombinant vector construction:
1) Linearize pHT43 by PCR;
2) Run 1% gel electrophoresis to verify the size of amplified fragments;
3) Extract DNA using the TIANgel Midi Purification Kit (Tiangen, Beijing, China);
4) Measure the purity and concentration of the DNA samples with a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA);
5) Use Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, Jiangsu, China) to construct the recombinant vectors pHT43-BsAiiA, pHT43-BsYtnP, pHT43-MtAiiM, pHT43-AtAttM, and pHT43-SsAhlS:
6) Transform the recombinant vectors into E. coli DH5α;
7) Screen on LB plates containing 50 μg/mL ampicillin;
8) Perform colony PCR for further screening;
9) Culture the positive transformants in liquid LB;
10) Extract plasmids using the TIANprep Rapid Mini Plasmid Kit (Tiangen, Beijing, China);
11) Confirm by DNA sequencing (GENEWIZ, Suzhou, Jiangsu, China).
Notes & Results:
Succeeded. Colony PCR results: Lanes 1-3: pHT43-BsAiiA; Lanes 4-6: pHT43-BsYtnP; Lanes 7-9: pHT43-MtAiiM; Lanes 10-12: pHT43-AtAttM; Lanes 13-15: pHT43-SsAhlS.
4. B. subtilis WB600 resistance test:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture B. subtilis WB600 from the stock culture in LB media;
3) Culture overnight at 30 ℃, 200 rpm;
4) Prepare solid LB media (Solarbio, Beijing, China);
5) Prepare solid LB plates containing 0, 11.3, 22.6, 34, 51, and 68 µg/mL chloramphenicol;
6) Spread 100 µL liquid culture from Step 3) on plates;
7) Culture at 30 ℃, observe colony growth.
Notes & Results:
51 µg/mL chloramphenicol was enough to totally inhibit the growth of B. subtilis WB600, which would be used in future transformant screening.
Week 3 (7.22-7.26)
5. Electroporation of B. subtilis WB600
1) Prepare 25 mL GMI solution in ddH2O with 2.28 g sorbitol and 1× LB, sterilizing using an autoclave;
2) Prepare 60 mL ETM solution in ddH2O with 5.45 g sorbitol, 5.45 g mannitol, and 6 mL glycerol, sterilizing using an autoclave;
3) Prepare 10 mL RM solution in ddH2O with 0.91 g sorbitol and 0.55 g mannitol, sterilizing using an autoclave;
4) Streak B. subtilis WB600 on an LB plate, incubate overnight at 30°C;
5) Pick a colony, culture overnight in 5 mL LB;
6) On day 2, inoculate 0.5 mL from step 5) into 25 mL GM in a flask. Incubate at 37°C, 200 rpm for about 4 hours until OD600 reaches 0.8;
7) Place the culture in an ice bath for 10 minutes. Centrifuge at 5000 g and 4°C for 5 min to collect the bacteria;
8) Resuspend the pellet with 15 mL cold ETM solution from step 2);
9) Repeat the wash four times;
10) Resuspend the pellet with 1 mL cold ETM solution;
11) Aliquot 60 µL into each 1.5 mL centrifuge tube. Store at -80°C;
12) Add 2 uL recombinant vectors;
13) Incubate at 4℃ for 5 min;
14) Load the mixture into a 1 mm cold MicroPulser Electroporation Cuvette (Bio-Rad, Hercules, CA, USA);
15) Shock once at 2.1 kV with MicroPulser Electroporator (Bio-Rad, Hercules, CA, USA);
16) Immediately add 1 mL warm RM solution from step 3);
17) Transfer into a 1.5 mL centrifuge tube, incubate at 37℃, 200 rpm for 3 hours;
18) Centrifuge at 5000 g for 5 min to collect the bacteria;
19) Screen on LB plates containing 51 ug/mL chloramphenicol. Culture overnight at 37℃;
Notes & Results:
We tried electroporation multiple times. We saw colonies grown on selective plates but failed to get any positive transformants. It is very interesting that B. subtilis seemed to get antibiotic resistance (we tried kanamycin and chloramphenicol) after electric shock, even in the negative control groups where no plasmid was added.
Some studies mentioned cellular stress could lead to antibiotic tolerance in bacteria (Nguyen et al., 2011; Poole, 2012; Yang et al., 2006). We assume electric shock caused stress responses in B. subtilis, enabling the growth on selective plates and leading to false positive clones in transformation.
Due to persistent failure, we turned to chemical transformation.
Week 4 (7.29-8.2)
6. Preparation of B. subtilis WB600 competent cells:
1) Prepare 100 mL 10× Spizizen salts in ddH2O with
14 g K2HPO4,
6 g KH2PO4,
2 g (NH4)2SO4,
1 g trisodium citrate, and
0.1 g MgSO4,
sterilizing using an autoclave;
2) Prepare 10 mL 10% yeast extract solution in ddH2O, sterilizing using an autoclave;
3) Prepare 10 mL 5% casein hydrolysate solution in ddH2O, sterilizing using an autoclave;
4) Prepare 10 mL 25% glucose solution in ddH2O, sterilizing using a 0.22 µm filter (BKMAM, Changde, Hunan, China);
5) Prepare 10 mL 0.1 M CaCl2 solution in ddH2O, sterilizing using a 0.22 µm filter (BKMAM, Changde, Hunan, China);
6) Prepare 10 mL 0.5 M MgCl2 solution in ddH2O, sterilizing using a 0.22 µm filter (BKMAM, Changde, Hunan, China);
7) Prepare 25 mL GMI solution by adding
2.5 mL 10× Spizizen salts,
250 uL 10% yeast extract,
100 uL 5% casein hydrolysate,
500 uL 25% glucose;
8) Prepare 100 mL GMII solution by adding
10 mL 10× Spizizen salts,
400 uL 10% yeast extract,
80 uL 5% casein hydrolysate,
2 mL 25% glucose,
500 uL 0.1 M CaCl2, and
500 uL 0.5 M MgCl2;
9) Prepare 10 mL resuspension solution by mixing 2 mL 50% sterile glycerol and 8 mL GMII;
10) Streak B. subtilis WB600 on an LB plate, incubate overnight at 30°C;
11) Pick a colony, culture overnight in 5 mL GMI at 30°C, 100 rpm;
12) On day 2, inoculate 2 mL from step 11) into 18 mL GMI in a flask. Incubate at 37°C, 200 rpm for 3.5 hours;
13) Inoculate 10 mL from step 12) into 90 mL GMII in a flask. Incubate at 37°C, 100 rpm for 1.5 hours;
14) Centrifuge at 5000 g for 10 min to collect the bacteria;
15) Resuspend the pellet with 10 mL resuspension solution from step 9);
16) Aliquot 500 µL into each 1.5 mL centrifuge tube. Store at -80°C. Each portion can be used for 5 transformations.
Notes & Results:
Succeeded. Confirmed in the following transformation.
Week 5 (8.5-8.9)
8. B. subtilis WB600 transformation:
1) Thaw competent cells at 37°C;
2) Aliquot 500 uL competent cells into 100 uL portions for each transformation;
3) Add 2 uL recombinant vectors;
4) Water bath at 37℃ for 30 min;
5) Incubate at 37℃, 200 rpm for 30 min;
6) Screen on LB plates containing 51 ug/mL chloramphenicol. Culture overnight at 37℃;
7) Perform colony PCR for further screening;
8) Culture the positive transformants in liquid LB;
Notes & Results:
Succeeded. Colony PCR results: Lane 1: pHT43-BsAiiA; Lane 2: pHT43-BsYtnP; Lane 3: pHT43-MtAiiM; Lane 4: pHT43- AsAhlD; Lane 5: pHT43-SsAhlS; Lane 6: pHT43-AtAttM.
9. IPTG Induction of engineered B. subtilis:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture the original, engineered, and commercial B. subtilis strains from the stock culture in LB media. Incubate at 37℃, 200 rpm;
3) When OD600 reaches 0.8, add IPTG to reach a final concentration of 1 mM;
4) Culture overnight at 30℃, 200 rpm;
Notes & Results:
Succeeded. Used in the following tests.
10. The zone of inhibition test:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture A. hydrophila ATCC 7966 from the stock culture in LB media;
3) Culture overnight at 30℃, 200 rpm;
4) Prepare solid LB media (Solarbio, Beijing, China);
5) Add 1/10 volume A. hydrophila culture from step 3) to molten LB gel (~50℃);
6) Mix thoroughly. Pour 15 mL of LB gel per 9 cm Petri dish;
7) Use a large pipette tip to punch 5 holes of 8.5 mm diameter into the agar;
8) Load 50 uL IPTG-induced B. subtilis culture into each hole;
9) Load 50 uL liquid LB as negative control;
10) Load 50 uL 50 ug/mL chloramphenicol as positive control;
11) Incubate overnight at 30℃;
12) Measure the diameter of the zones of inhibition.
Notes & Results:
All operations involving A. hydrophila (Risk Group 2) were carried out in Class II biosafety cabinets in a BSL-2 laboratory.
Results showed that all the strains were unable to inhibit the growth of A. hydrophila. We were surprised at first and went for literature research. It turned out that this result was reasonable since quorum sensing primarily regulates virulence factors rather than bacterial growth. This is also consistent with some previous studies on A. hydrophila (Chen et al., 2020; Chu et al., 2014).
Since direct growth inhibition of A. hydrophila was not feasible, we shifted our focus to testing the degradation of AHL and the inhibition of virulence factors of A. hydrophila using our engineered B. subtilis.
Week 6 (8.12-8.16)
11. Assessment of engineered B. subtilis for synthetic AHL degradation:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture Chromobacterium subtsugae CV026 from the stock culture in LB media;
3) Culture overnight at 30℃, 200 rpm;
4) Prepare solid LB media (Solarbio, Beijing, China);
5) Add 1/10 volume CV026 culture from step 3) to molten LB gel (~50℃);
6) Also, add N-butanoyl-L-homoserine lactone (C4-HSL) (Macklin, Shanghai, China) to molten LB gel to reach a 20 uM final concentration;
7) Mix thoroughly. Pour 15 mL of LB gel per 9 cm Petri dish;
8) Use a large pipette tip to punch 4 holes of 8.5 mm diameter into the agar;
9) Load 50 uL IPTG-induced B. subtilis culture into each hole;
10) Load 50 uL liquid LB as negative control;
11) Incubate overnight at 30℃;
12) Measure the diameter of the zone without the purple color;
13) For mixed tests, mix two B. subtilis cultures in a 1:1 ratio and repeat the test.
Notes & Results:
C4-HSL is the AHL synthesized and sensed by Aeromonas hydrophila. C. subtsugae CV026 was used as the AHL biosensor in this experiment, where exogenous AHL triggers the synthesis of purple pigment.
Results showed that all the strains degraded synthetic C4-HSL, with the strain expressing BsAiiA exhibiting the highest level of degradation.
12. Assessment of engineered B. subtilis for natural AHL degradation:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture C. subtsugae CV026 from the stock culture in LB media;
3) Culture overnight at 30℃, 200 rpm;
4) Subculture Aeromonas hydrophila ATCC 7966 from the stock culture in LB media;
5) Culture overnight at 30℃, 200 rpm;
6) Prepare solid LB media (Solarbio, Beijing, China);
7) Add 1/10 volume CV026 culture from step 3) to molten LB gel (~50℃);
8) Mix thoroughly. Pour 15 mL of LB gel per 9 cm Petri dish;
9) Use a large pipette tip to punch 4 holes of 8.5 mm diameter into the agar;
10) Load 25 uL IPTG-induced B. subtilis culture mixed with 25 uL A. hydrophila culture from step 5) into each hole;
11) Load 50 uL liquid LB as negative control;
12) Load 50 uL A. hydrophila culture as positive control;
13) Load 50 uL 1 mM C4-HSL solution as positive control;
14) Incubate overnight at 30℃;
15) Measure the diameter of the purple zone;
16) For mixed tests, mix two B. subtilis cultures in a 1:1 ratio and repeat the test.
Notes & Results:
All operations involving A. hydrophila (Risk Group 2) were carried out in Class II biosafety cabinets in a BSL-2 laboratory.
Results showed that, except for AtAttM, all the engineered strains were able to completely degrade C4-HSL synthesized by A. hydrophila in this experiment.
13. Assessment of engineered B. subtilis for reducing A. hydrophila biofilm formation:
1) Prepare liquid LB media (Solarbio, Beijing, China);
2) Subculture A. hydrophila ATCC 7966 from the stock culture in LB media;
3) Culture overnight at 30℃, 200 rpm;
4) Acquire the supernatants of IPTG-induced B. subtilis cultures by centrifugation;
5) For each experimental group, mix 200 uL culture supernatant and 200 uL fresh LB in a 1.5 mL tube;
6) Mix 200 uL water and 200 uL fresh LB as the control group;
7) Inoculate 40 uL A. hydrophila culture from step 3) into each tube;
8) Incubate under static conditions for 48 hours at 30℃;
9) On day 3, measure the OD600s with an automated ELISA analyzer and 96-well plates;
10) Carefully remove the cultures without disturbing the biofilm on the tube walls;
11) Wash tubes twice with 1000 uL water. Air dry the tubes;
12) Dye the biofilms with 500 uL 1% crystal violet (dissolved in ethanol) for 10 min;
13) Carefully remove the cultures without disturbing the biofilm on the tube walls;
14) Wash tubes three times with 1000 uL water. Air dry the tubes;
15) Add 1 mL of ethanol, then scrape the biofilms using pipette tips. Vortex until a homogeneous purple solution is obtained;
16) Measure the OD570s with an automated ELISA analyzer and 96-well plates;
17) For mixed tests, mix two B. subtilis cultures in a 1:1 ratio and repeat the test.
Notes & Results:
All operations involving A. hydrophila (Risk Group 2) were carried out in Class II biosafety cabinets in a BSL-2 laboratory.
Results showed that all the strains helped to reduce the biofilm of A. hydrophila, with the strain expressing BsAiiA exhibiting the highest level of reduction.
14. Assessment of engineered B. subtilis for reducing A. hydrophila extracellular proteases:
1) Collect the cultures from the biofilm test in step 10);
2) Collect the supernatants by centrifugation;
3) Measure the activities of proteases in the supernatants using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China);
4) For mixed tests, mix two B. subtilis cultures in a 1:1 ratio and repeat the test.
Notes & Results:
All operations involving A. hydrophila (Risk Group 2) were carried out in Class II biosafety cabinets in a BSL-2 laboratory.
In this test, although our intention was to measure the changes in protease levels of A. hydrophila, since we used the supernatants of the B. subtilis cultures, we could not distinguish whether the proteases originated from B. subtilis or A. hydrophila. Consequently, the commercial B. subtilis group exhibited extremely high levels of protease activity compared to other groups, suggesting that most of the proteases were from B. subtilis itself rather than A. hydrophila.
In contrast, our engineered strains are derived from the WB600 strain, which has six major protease genes knocked out, resulting in minimal endogenous protease activity. This low baseline level makes the test feasible, as most of the proteases measured should originate from A. hydrophila.
Results showed that all the strains helped to reduce the extracellular protease activities of A. hydrophila, with the strain expressing BsAiiA exhibiting the highest level of reduction.
References
Chen, B., Peng, M., Tong, W., Zhang, Q., & Song, Z. (2020). The Quorum Quenching Bacterium Bacillus licheniformis T-1 Protects Zebrafish against Aeromonas hydrophila Infection. Probiotics Antimicrob Proteins, 12(1), 160-171. https://doi.org/10.1007/s12602-018-9495-7
Chu, W., Zhou, S., Zhu, W., & Zhuang, X. (2014). Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection. Sci Rep, 4, 5446. https://doi.org/10.1038/srep05446
Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B. E., & Singh, P. K. (2011). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science, 334(6058), 982-986. https://doi.org/10.1126/science.1211037
Poole, K. (2012). Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria. Trends Microbiol, 20(5), 227-234. https://doi.org/10.1016/j.tim.2012.02.004
Yang, S., Lopez, C. R., & Zechiedrich, E. L. (2006). Quorum sensing and multidrug transporters in Escherichia coli. Proc Natl Acad Sci U S A, 103(7), 2386-2391. https://doi.org/10.1073/pnas.0502890102