Team:NDNF China/Experiments
Plasmid Construction
Hydrogel Preparation
1. Sterilize 5% sodium alginate at 115°C for 15min, then mix with the bacteria liquid in a volume ratio of 2:1.
2. Form the core using syringes then soak in 5% calcium chloride solution.
3. Rinse the core and set aside to dry off before coating starts.
4. Sterilize 10% sodium alginate at 115℃ for 15min, mix with Acr-Bis solution of different concentration at ratio 1:4 (0.046% ammonium persulfate, 2000:1 tetramethylethylenediamine (TEMED) were added to the Acr-Bis solution in advance)
5. Preload the alginate and Acr-bis mixture into a mould, and lodge in the previously made core.
6. Soak in MES + Crosslinkers
a. Cross-linking system: 0.3M calcium chloride, 0.00125% of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide 1-ethyl-(3-dimethylaminopropyl) carbodiimide Amine (EDC), 0.000375% N-hydroxysuccinimide N-hydroxysuccinimide, and 0.00075% adipic acid dihydrazide (ADH) mixed, dissolved in MES buffer
b. MES buffer: 2-N-morpholino-ethanesulfonic acid MES 9.76g and NaCl 14.61g are mixed and dissolved, and the volume is adjusted to 500ml PH6.0
Tests
Escape prevention
1. Add 2 ml of LB medium plus antibiotics and CaCl2 (20mM) to each well of a 24-well plate.
2. Put the prepared hydrogel beads into the 24-well plate (one bead in each well).
3. Take 150 μL of medium and take photos at regular intervals. Use the absorbance OD600 for detection, then plot the escape curve.
Colour expression
1. Encapsulate bacteria containing certain genetic circuit into hydrogel beads (procedures are mentioned above).
2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).
3. Beads containing engineered bacteria are incubated in LB plus antibiotics and arabinose at 37 °C.
4. Observe the color of each bead.
Mercury sensing
[bacteria]1. Cells are grown overnight at first, and are transformed into M9 (150 μL, 96-well plate) plus antibiotics and mercury ion (10μM) at the ratio of 1:100. Use the same amount of mercury-sensing bacteria and M9 medium plus antibiotics for serial dilution.
2. 8 h of incubation at 37 °C
3. Fluorecence tests using plate reader with the range of wavelength 485/535.
4. Bacteria liquid concentration tests using plate reader with the wavelength of 600.
5. Plot the fluorescence testing curve.
[in beads]1. Encapsulate bacteria containing mercury sensing genetic circuit into hydrogel beads.
2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h.. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).
3. Beads containing engineered bacteria are incubated in LB plus antibiotics and mercury ion (10μM) at 37 °C for 8 h.
4. Test results can be analysed by fluorescence microplate assays or fluorescence microscope.
a. Fluorescence microplate assays:
i. Remove the shell of hydrogel beads using a razor blade and a tweezer.
ii. Place the hydrogel beads in a vitreous bead beater, add 0.5 mL of filtered, sterilized PBS. Homogenize hydrogel beads using bead beater.
iii. After centrifugation at 300g for 5min, 150 µL of supernatant was taken into 96-well plates, and microplate reader is used to test fluorescence in the wavelength range of 485/535 nm.
b. Fluorescence microscope: Slice the bead with a sharp razor blade at thickness of roughly 0.5 mm. The sliced sample was then imaged with a fluorescence microscope with excitation wavelength at 485 nm and emission wavelength at 535 nm.
Caffeine sensing
[bacteria]1. Cells are grown overnight at first, and are transformed into M9 (150 μL, 96-well plate) plus antibiotics, arabinose and caffeine molecules (100μM) at the ratio of 1:100. Use the same amount of caffeine-sensing bacteria and M9 medium plus antibiotics for serial 2-fold dilution.
2. 8 h of incubation at 25 °C
3. Fluorecence tests using plate reader with the range of wavelength 535/595.
4. Bacteria liquid concentration tests using plate reader with the wavelength of 600.
5. Plot the fluorescence testing curve.
[in beads]1. Encapsulate bacteria containing caffeine sensing genetic circuit into hydrogel beads.
2. Each bead is placed in a well on a 24-well plate and incubate at 37 °C for 12 h.. Any beads that show bacterial growth in the surrounding media should be discarded. (Due to the toxicity of the Crosslinker, the bacteria needed to be incubated before testing).
3. Beads containing engineered bacteria are incubated in LB plus antibiotics, arabinose (10 mM), and caffeine molecules (100 µM) at 25 °C for 12 h.
4. Test results can be analysed by fluorescence microplate assays or fluorescence microscope.
a. Fluorescence microplate assays:
i. Remove the shell of hydrogel beads using a razor blade and a tweezer.
ii. Place the hydrogel beads in a vitreous bead beater, add 0.5 mL of filtered, sterilized PBS. Homogenize hydrogel beads using bead beater.
iii. After centrifugation at 300g for 5min, 150 µL of supernatant was taken into 96-well plates, and microplate reader is used to test fluorescence in the wavelength range of 535/595 nm.
b. Fluorescence microscope: Slice the bead with a sharp razor blade at thickness of roughly 0.5 mm. The sliced sample was then imaged with a fluorescence microscope with excitation wavelength at 535 nm and emission wavelength at 595 nm.
Stress resistance
Heat resistance
[in beads]1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.
2. Group A - suitable conditiona. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 2 h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
3. Group B - thermal stimulusa. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 90 ℃ for 2 h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
d. Biological replication twice.
[bacteria]1. Group A - suitable condition
a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 2 h.
b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
2. Group B - thermal stimulusa. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 90℃ for 2 h.
b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
c. Biological replication twice.
Acid resistance
[in beads]1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.
2. Group A - suitable conditiona. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 1 h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
3. Group B - acid stimulusa. Place two hydrogel beads in two 1.5 mL electropolished tubes. Add 500 μL LB media plus antibiotics with pH values of 1.5 and 2.5 to each tube, and incubate at 37 ℃ for 1h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
d. Biological replication twice for each pH condition.
[bacteria]1. Group A - suitable condition
a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 1 h.
b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
2. Group B - acid stimulusa. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media plus antibiotics with pH values of 1.5 and 2.5, incubate at 37℃ for 1 h.
b. Use 0.5 ml filtered, sterilized PBS to dissolve dry bacteria. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
c. Biological replication twice.
Alkali resistance
[in beads]1. Hydrogel beads are incubated in a 24-well plate at 37 ℃ for 12 h for bacteria strains inside to grow.
2. Group A - suitable conditiona. Place a hydrogel bead in a 1.5 mL electropolished tube. Add 500 μL LB media plus antibiotics, and incubate at 37 ℃ for 1 h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
3. Group B - alkali stimulusa. Place two hydrogel beads in two 1.5 mL electropolished tubes. Add 500 μL LB media plus antibiotics with pH values of 10 and 11 to each tube, and incubate at 37 ℃ for 1h.
b. Take out the hydrogel bead after incubation and remove the shell with a razor blade and a tweezer. Place the core of the bead in a 0.5 mL vitreous bead beater, add 0.5 mL of filtered, sterilized PBS, and homogenize the bead using the bead beater.
c. Add 0.2 ml homogenate into a 96-well plate, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
d. Biological replication twice for each pH condition.
[bacteria]1. Group A - suitable condition
a. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media with antibiotics, incubate at 37℃ for 1 h.
b. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
2. Group B - alkali stimulusa. Take 20 μL bacteria liquid incubated overnight, add 0.5 ml LB media plus antibiotics with pH values of 10 and 11, incubate at 37℃ for 1 h.
b. Use 0.5 ml filtered, sterilized PBS to dissolve dry bacteria. Add 0.2 ml bacteria liquid into a 96-well plate after incubation, serial 10-fold dilution is used. Assume the concentration of homogenate is 1, take 8 µL of solution with the concentration of 1~10^(-5). Plate count using plates with antibiotics.
c. Biological replication twice.
L-dopa expression
1. Pick a single colony from streaked microbes on the plate, and cultivate at 37°C overnight.
2. Encapsulate bacteria containing the caffeine circuit into hydrogel beads, as well as beads enclosing unmodified strains for control.
3. Incubate the beads in LB plus antibiotics and caffeine molecules (100 mM) at 37 °C for 24h. The liquid medium should also contain 4.5mM of tyrosine and 1mM of IPTG.
4. Transfer 180μl of the liquid medium from each well into a 96-well plate individually, then add 20 µl of 100 mM NaIO3, leave for half an hour under room temperature to react.
5. Compare with L-dopa colour standard.
Catalytic enzymes expression
1. Pick a single colony on the plate and place it in the corresponding resistant 5ml medium, and cultivate overnight at 37°C.
2. Transfer the overnight cultured bacterial solution 1:100 into 8ml M9 medium containing the corresponding antibiotic arabinose (10 mM) and caffeine molecules (100 µM), induce overnight at 30°C.
3. Collect the bacterial cells. Centrifuge the induced bacterial solution at 4℃ 5000rpm for 5min.
4. Wash the bacteria 2-3 times with 5ml PBS.
5. Resuspend the bacteria in 1ml filtered sterile PBS buffer.
6. 150w ultrasound to break the bacteria on ice until the bacteria are completely broken.
7. Centrifuge at 20000g for 20min, take the supernatant and filter it with a 0.45μm filter membrane.
8. The filtrate and protein electrophoresis buffer are mixed evenly at a ratio of 4:1 and heated at 90°C for 5 minutes.
9. SDS PAGE
Toxin protein expression
1. Pick a single colony with arabinose-induced toxin expression gene circuit from the plate and culture it overnight.
2. Transfer the 1:100 bacterial solution cultured overnight to 5ml of 10mM arabinose medium containing the corresponding antibiotic, and culture it overnight at 37°C in the corresponding antibiotic-free arabinose medium.
3. Dilute the overnight cultured bacteria in a 10-fold gradient in a 96-well plate, record the concentration of the undiluted bacteria as 1, and take 8 microliters each of the negative fifth power of the concentration of 1-10. Gradient dot plate counting.
4. Calculate killing efficiency.
Competent Preparation
1. Streak and culture top 10 strain with pCas plasmid overnight on kana resistant LB plate (the seeds are from competent cells).
2. Pick a single colony and culture it overnight at 30°C in 4ml LB medium.
3. The flask is cultured with shaking at 30°C until the OD600 reaches 0.4-0.5. The time required for this process depends on the type of strain. Add arabinose with a concentration of 10mM one hour before collecting the bacteria.
4. Usually after 2.5h-4h, three flasks are quickly placed on ice, shaken vigorously, and the temperature is quickly cooled.
5. For the subsequent processes: Try to carry out at low temperature (add 40g NaCl in the ice box).
6. Transfer the medium to a pre-cooled centrifuge tube at 4°C 4500rpm and centrifuge for 10 minutes, fully discard the supernatant and add 2/3 volume of pre-cooled CaCl2-MgCl2 to each tube.
7. Mix the solution, resuspend the cells, ice bath for 10 minutes at 4°C, centrifuge at 4500 rpm for 10 minutes, fully discard the supernatant.
8. Add 1/25 volume of pre-cooled 0.1M CaCl2 solution to each tube, resuspend the cells, ice bath for 10 minutes, add 7% to each tube Pre-cooled DMSO, ice bath for 10 minutes.
9. Distribute into 1.5ml EP tubes, each EP tube containing 100ul resuspension solution, and quickly freeze the cells with liquid nitrogen, then put them in a -80°C refrigerator.
Barcode addition
1. Transform pCas plasmid into top10 strain, kana resistant plate. 30℃ culture screening
2. Pick out positive clones and prepare competent clones according to the method in the previous section.
3. The chemical transformation method transforms the plasmid pTargetT containing homologous fragments and barcode into competent, and after 1 hour of recovery at 30°C, screen with kana+amp plate.
4. Verify the clones after overnight culture to verify the success of gene editing.
5. Pick a single colony that has been successfully edited in LB liquid medium (kana resistance), add 0.5mM IPTG and incubate for 8-20 hours, streak out the single bacteria, and pick single colonies and inoculate them respectively for kana resistance And kana+amp resistant medium, verify the elimination of pTargetT plasmid.
6. After verifying that the pTargetT plasmid is successfully eliminated, transfer the bacterial solution to non-resistant LB liquid medium and cultivate at 40°C to eliminate pCas.
7. Streak out the single bacteria and pick a single colony, respectively inoculate them in non-resistant and kana resistant medium to verify the elimination of pCas plasmid.
RPA
Use colloidal gold test strip DNA rapid amplification Kit at room temperature
Put the solution below into an environment with 39℃ for 8mins.
Barcode testing
1. Assemble the reaction at room temperature in the order above (table on the upper right):
2. Pre-incubate for 10 minutes at 25⁰C.
3. Add 3 µl of 30 nM substrate DNA (30 µl final volume).
4. Mix thoroughly and pulse-spin in a microfuge.
5. Incubate at 37°C for 10 minutes.
6. Incubate at room temperature for 10 minutes.
7. Proceed with analysis.
