We obtained bacteria samples from the murals at Dr. Wu Fasi's laboratory. The bacteria samples were inoculated onto LB solid culture medium plates and incubated at 37°C overnight. Select individual colonies for 16S fragment PCR amplification. The amplified products are sent for sequencing and then subjected to BLAST comparison.
We obtained fungal samples from the murals at Dr. Wu Fa Shi's laboratory. The fungal samples were inoculated onto PDA solid culture medium plates and incubated at 30°C for 7 days. Spores were collected. The spore suspension was diluted and streaked for plating, and single colony mycelia were picked for testing. The identified single colonies were purified and cultured, and the spores were collected for future use.
The recombinant plasmid was constructed by inserting BBa_25XI83ND and BBa_259HIEMB into pET-30a(+) and transformed into Escherichia coli BL21(DE3) cells. Expression of BBa_25XI83ND and BBa_259HIEMB was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) until the optical density at 600 nm (OD600) reached 0.6 at 37°C, followed by incubation at 16°C for 16-20 hours.
The isolated bacteria were inoculated onto LB solid culture medium plates and cultured at 37℃ overnight. Make holes on the culture medium, add 200 μL of the induced BL21(DE3)-pET-30a(+) - BBa_25XI83ND bacterial solution into the holes, and incubate at 37℃ overnight. Then measure the size of the inhibition zone.
The spore suspension was spread on a PDA solid culture medium plate and incubated at 30℃ for 4 days until visible mycelium was formed. Holes were drilled on the culture medium, and 200 μL of the induced-expressed BL21(DE3)---pET-30a(+)---BBa_259HIEMB bacterial solution was added to each hole. The bacteria were incubated at 30℃ for 2 days, and the size of the inhibition zone was observed and measured.
In order to test the survival of our engineered bacteria on the mural, we obtained simulated mural samples from Dr. Wu Fasi's laboratory.
The selected fungi Scopulariopsis brumptii, Scopulariopsis Hibernica, Aspergillus versicolor and bacteria Bacillus sp. (in: Bacteria) strain 2YHBJ3, Bacillus sp. DYJL12, Bacillus amyloliquefaciens strain T8M7, Priestia megaterium strain 38-Y92, Priestia megaterium strain SEM47, Priestia sp. strain W-11 were centrifuged and mixed in equal proportions. The cells were resuspended in an equal mixture of PDA and LB medium. 10 ml of the bacterial suspension was placed in a petri dish. A simulated mural was placed upside down in the dish to absorb the bacterial suspension. The dish was sealed and incubated upright at 30°C for 5 days. Then, 5 ml of the engineered antimicrobial bacteria BL21- BBa_25XI83ND / BBa_259HIEMB (OD = 1.0) was sprayed onto the simulated mural. After one day of incubation, the mural was stained with Calcein-AM/PI to analyze the inhibitory effect of the engineered bacteria on the microorganisms on the mural.
The recombinant plasmid was constructed by inserting BBa_256VH953 and BBa_25OTZVL1 into pET-30a(+) and transformed into Escherichia coli BL21(DE3) cells. Expression of BBa_25XI83ND and BBa_259HIEMB was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) until the optical density at 600 nm (OD600) reached 0.6 at 37°C, followed by incubation at 16°C for 16-20 hours.
Carotenoids were first mixed with 1 ml of Tween-80, and then ultrasonication was used to make the solution into an emulsion. Finally, the Tween was diluted with water to 0.5%, and the concentration of carotenoids was 20 mg/L. A 20% inoculum was used for degradation for 12 hours. The absorbance was measured at 450 nm using a UV-visible spectrometer. The absorbance values were compared with those of the control group without adding the engineered bacteria and without inducing the expression of the engineered bacteria.
Melanin dissolves in water and is prepared at a concentration of 50 mg/L. The strain is washed three times with PBS and inoculated at a 20% inoculation volume. The degradation process lasts for 12 hours. The decolorization efficiency of melanin is measured using a spectrophotometer at 540 nm. The absorbance values are compared with those of the control group without adding the engineered bacteria and without inducing the expression of the engineered bacteria.
Take 200 μL of competent cells, add 50 ng of recombinant plasmid DNA to the tube, gently rotate to mix the contents, and incubate on ice for 30 minutes. Place the tube in a 42°C water bath for 1.5 minutes (without shaking). Then quickly cool the tube in ice water for 2 minutes. Add 1000 μL of LB liquid medium to each tube, and incubate in a 37°C constant temperature incubator for 45 minutes by shaking.
Take 100 μL of each well-fermented bacterial solution and apply it to the LB medium containing kanamycin using glass beads to spread the bacterial solution.
The coated plates were placed in a 37℃ constant temperature incubator and inverted for overnight cultivation for 20 hours. The results were observed the next day and recorded.
After overnight culture, pure single colonies were taken from the plates and cultured in LB liquid medium to obtain bacterial solutions of different species. These solutions were then incubated in a 37°C constant temperature incubator overnight and stored in a 4°C refrigerator.
Take 2 μL of the successfully cultivated strain for the strain PCR. The reaction system consists of 25 μL of Taq r, 2 μL each of forward and reverse primers, 2 μL of the bacterial solution, and 19 μL of dd H2O, totaling 50 μL. The PCR instrument parameters are set as follows: annealing at 95℃ for 30 seconds, denaturation at 60℃ for 0 seconds, extension at 72℃ for 10 seconds, with 30 cycles. After the PCR is completed, take the sample and perform agarose gel electrophoresis, then observe the phenomenon using ultraviolet light.
The transformed BL21-pSB1A3-test(Amp+) was taken out from the cryotube and activated in LB medium containing Amp for two hours. Prepare M9 media with pH values of 4, 4.25, 4.75, 5, 5.5, 6, 6.5, and 7; in a 96-well plate, add 12 wells of each pH M9 medium, with 200 μl in each well , (assuming one row for each pH) add 25 μl of the activated bacterial solution to holes A to F, 25 μl of LB medium to holes G to I, and 25 μl of the untransformed E. coli solution to holes J to L (with the OD value of the activated engineered bacteria being the same); set the spectrophotometer to 30℃, and measure the OD600, 507nm fluorescence, and 610nm fluorescence of all wells every ten minutes.
We will subtract the data corresponding to the blank Escherichia coli from the obtained data to eliminate the background influence. All the original data will be divided by the measured OD value to perform normalization on the data.
For every 100 mL of LB medium, add 1 g NaCl, 1 g tryptone, 1 g yeast extract, and 2 g agar powder. Sterilize by autoclaving at 121°C for 20 minutes. Prepare a 20% arabinose aqueous solution as the stock solution. Add the required amount of stock solution to achieve the desired concentration before pouring the plates.
Retrieve competent E. coli cells stored at -80°C and thaw on ice for 15 minutes. Add 1 μL of the plasmid to be transformed and incubate on ice for 15 minutes. Heat-shock the cells in a 42°C water bath for 1 minute and 30 seconds, then place on ice for 2 minutes. Add 900 μL of plain LB liquid medium and incubate at 37°C with shaking at 180 rpm for 1 hour. Spread 100 μL of the bacterial suspension onto solid medium using sterile glass beads.
1. Cloning PCR System
2. Gel Extraction of Cloning Product
(1) Perform agarose gel electrophoresis on the fusion PCR product and excise the band of appropriate size (approximately 3500 bp) for gel extraction.
(2) Place the gel slice in a 2 mL centrifuge tube, add an appropriate amount of Binding Buffer based on the gel size, and incubate in a 50-60°C water bath until the gel is completely dissolved, mixing by inversion every 1-2 minutes.
(3) Transfer the solution to a spin column and centrifuge at 12,000 ×g for 1 minute.
(4) Discard the flow-through, add 300 μL Binding Buffer, and centrifuge at 12,000 ×g for 1 minute.
(5) Add 700 μL of buffer (pre-mixed with absolute ethanol) and centrifuge at 12,000 ×g for 1 minute.
(6) Repeat step (5).
(7) Discard the flow-through and centrifuge at 12,000 ×g for 2 minutes.
(8) Open the lid of the spin column and place it in a clean 1.5 mL centrifuge tube. Air-dry for 10 minutes.
(9) Add 15-30 μL of Elution Buffer or ddH₂O and let it stand for 5 minutes.
(10) Centrifuge at 12,000×g for 2 minutes to obtain the DNA product.
3. Digestion of pUC19 Vector and Cloning Product
Digestion System:
4. Gel Extraction of Digested Products
5. Ligation of Vector and Fragment
(1) Ligation System:
(2) Incubate at 16°C for ≥8 hours.
6. Transformation of Ligation Product
(1) Thaw competent E. coli cells on ice for 15 minutes.
(2) Add the ligation product and incubate on ice for 15 minutes.
(3) Heat-shock in a 42°C water bath for 1 minute and 30 seconds.
(4) Place on ice for 2 minutes.
(5) Add 900 μL of LB liquid medium containing 1% arabinose.
(6) Incubate at 37°C with shaking at 180 rpm for 1 hour.
(7) Spread 100 μL onto LB solid medium with 1% arabinose and without arabinose, then incubate at 37°C.
7. Colony PCR
(1) Pick single E. coli colonies (non-red in color) from the medium containing 1% arabinose and resuspend in 30 μL ddH₂O in a 0.2 mL centrifuge tube.
(2) Colony PCR System:
(3) PCR Program:
(4) Analyze 4 μL of PCR product by agarose gel electrophoresis.
(5) Inoculate colonies with the target band into 5 mL LB medium and culture at 37°C with shaking at 180 rpm.
(6) Send bacterial culture or extracted plasmids for sequencing using M13F and M13R universal primers for bidirectional sequencing.
8. Transformation and Induced Expression of the Nuclease Gene
Induce expression with 50 mM IPTG.
1. PCR System
2. PCR Program
3. Product Purification
(1) Analyze 4 μL of PCR product by agarose gel electrophoresis. Confirm a single band of the correct size and purify the product using a gel extraction kit.
(2) Take an empty spin column and centrifuge tube, add 300 μL Binding Buffer, then add the PCR product and mix by pipetting.
(3) Centrifuge at 12,000 ×g for 1 minute.
(4) Discard the flow-through, add 300 μL Binding Buffer, and centrifuge at 12,000 ×g for 1 minute.
(5) Add 700 μL of buffer (pre-mixed with absolute ethanol) and centrifuge at 12,000 ×g for 1 minute.
(6) Repeat step (5).
(7) Discard the flow-through and centrifuge at 12,000 ×g for 2 minutes.
(8) Open the lid of the spin column and place it in a clean 1.5 mL centrifuge tube. Air-dry for 10 minutes.
(9) Add 15-30 μL of Elution Buffer or ddH₂O and let it stand for 5 minutes.
(10) Centrifuge at 12,000 ×g for 2 minutes to obtain the DNA product.
4. Digestion of PCR Product and pSB1A3 Vector
(1) Digestion System:
(2) Incubate at 37°C for 30 minutes.
5. Gel Extraction of Digested Products
The regulatory part of the suicide module is approximately 2300 bp, and the pSB1A3 digested product should be around 2000 bp.
6. Ligation of PCR Product and Vector Fragment
(1) Ligation System:
(2) Incubate at 16°C for ≥8 hours.
7. Transformation into E. coli DH5α and Plating on LB Solid Medium
8. Colony PCR of Single Colonies
(1) Colony PCR System:
(2) Analyze 4 μL of PCR product by agarose gel electrophoresis.
(3) Inoculate colonies with the target band into 5 mL LB medium and culture at 37°C with shaking at 180 rpm.
9. Extraction of Plasmid DNA Using a Miniprep Kit
10. Digestion of mazF Fragment and Vector Fragment
11. Gel Extraction of Digested Products
The mazF fragment is approximately 300 bp.
12. Ligation of mazF and Vector Fragment
13. Transformation of Ligation Product into E. coli
Plate on LB solid medium with 1% arabinose and without arabinose.