ROS-inducible catalase experiments

Experimental Overview

Our primary experiments focus on the engineering of a ROS-inducible catalase system designed to effectively neutralise reactive oxygen species within the coral microbiome and thereby protecting them from bleaching. This system is intended to be optimized for both control - selecting the best promoter that elicits the most effective response to ROS - and neutralization - identifying the most effective catalase for this application.

To facilitate the assembly and optimization of our system, we utilised Golden Gate cloning, allowing us to efficiently combine various components as needed. This modular approach enhanced our ability to iteratively refine our system.

For our promoter selection, we chose:

• OxyR/KatG: the native promoter for KatG in E. coli.
• OxyR/TrxCp: another E. coli promoter known for its ROS sensitivity.
• SoxR/SoxS: a superoxide E. coli promoter that may facilitate multi-ROS activity.

For the catalases, we selected:

• KatG from E. coli.
• KatG from Mycobacterium tuberculosis (strain H37Rv)
• Kat from Geobacillus sp. CHB1.

To validate our system, initial laboratory testing was conducted in E. coli, which provided a controlled environment to assess the effectiveness of these components before progressing to more complex systems in the coral microbiome.

Plasmid construction

All the reagents and gBlocks used for the construction of each clone are detailed in the Cloning Guide. Constructs 1-5 were used to make 6-8, which served as biosensors. In these constructs, RFP was regulated by our ROS-sensitive promoters to measure their efficacy under increasing concentrations of hydrogen peroxide. Constructs 9-11 were designed to express our 3 catalase genes, using a rhamnose promoter which acted as an ON/OFF switch to provide simple negative controls.

• Prepared the necessary reagents (see cloning guide) and gBlocks for golden gate cloning
• Combined DNA fragments and reaction mixture in a PCR tube.
• Incubated the reaction mixture in the thermocycler, cycling between digestion and ligation.

1. Thawed 100uL E coli DL21 cells and glycerol on ice.
2. Thawed 100uL E coli DL21 cells and glycerol on ice.
3. Combined 100uL cells and 1uL golden gate reaction mixture in a cuvette for each construct
4. Prepared 900uL of Super Optimal Broth for each reaction
5. For each construct, applied an electric pulse and immediately added the mixture to the SOB.
6. Recovered in the SOB for 1 hour at 37°C.
7. Spread 100uL of transformed cells onto plates containing selective media (see Notebook) and incubated overnight at 37 degrees.

1. Selected individual colonies from the transformation plates using a sterile pipette tip.
2. Transferred each colony into LB broth with selective media (see Notebook).
3. Incubated the cultures overnight at 37°C on a shaking incubator.

1. Pelleted the cells by centrifugation at 13,000 rpm for 1 minute.
2. Discarded the supernatant and resuspended the cell pellet in 250 µL of resuspension buffer.
3. Added 250 µL of lysis buffer to the resuspended cells and mixed gently by inversion.
4. Added 350 µL of neutralisation buffer and mixed gently by inversion.
5. Centrifuged the mixture for 10 minutes at 13,000 rpm to pellet cell debris.
6. Transferred the supernatant to a spin column and centrifuged for 1 minute.
7. Washed the column with 500 µL of wash buffer and centrifuged for 1 minute.
8. Repeated the wash step with another 500 µL of wash buffer, followed by centrifugation.
9. Eluted the plasmid DNA by adding 50 µL of elution buffer to the center of the column and centrifuged for 1 minute.
10. Measured the concentration of plasmid DNA using Nanodrop

1. Added 4.5 uL of each plasmid, 1 uL cutsmart buffer, 4 uL H2O and 0.5 uL of NdeI restriction enzymes into microtubes.
2. Incubated in a thermocycler overnight.

Hydrogen peroxide experiments

1. For each construct, transferred 300uL starter culture and 20mL LB with spec and carb to new Falcon tubes.
2. Measured the optical density to ensure it was in the range of 0.05-0.08.
3. Prepared hydrogen peroxide solutions in Round-Bottom Polypropylene Test Tubes to achieve final concentrations of 0mM, 5mM, 10mM, 20mM and 30mM, given a final volume of 2mL and from a 1M stock. For each concentration, there should be one tube for every construct, labelled with both the plasmids (eg 6+9) and the H2O2 concentration (eg 0mM).
4. For the negative controls, added 2mL of diluted culture without rhamnose to corresponding tubes (constructs 6+9 and 8+9)
5. Added 500uL 20% L-rhamnose to each 20mL dilution to induce catalase plasmid expression.
6. Transferred 2mL of each culture to corresponding test tubes.
7. Incubated at 37 degrees on a shaking incubator (exact times described in Notebook)

1. Added 100uL of each culture incubated with hydrogen peroxide to a 96-well plate.
2. Measured absorbance at 600 nm using Infinite M Plex plate reader.
3. Measured fluorescence with excitation at 570 nm and emission at 610 nm.

1. Transferred 100uL of each starter culture and 5mL LB with spec and carb to new 50mL Falcon tubes
2. Incubated on a shaking incubator at 37 degrees for 2 hours, until the cultures reached 0.7-1 OD600. At this stage, the cells should be in their log phase.
3. Back-diluted the cultures to an OD600 of 0.2 by diluting them in LB, bringing the total volume to 7.5 mL in new 15 mL Falcon tubes.
4. Prepared hydrogen peroxide solutions in 1.5mL Eppendorf Tubes to achieve final concentrations of 0mM, 0.25mM, 0.5mM, 1mM and 2.5mM and 5mM, given a final volume of 1mL. For each concentration, we used one tube for every construct.
5. Added 1 mL of diluted culture (without L-rhamnose) to the corresponding test tubes for constructs 6+9 and 8+9, to act as negative controls.
6. For all cultures, including constructs 6+9 and 8+9, added 190 µL of 20% L-rhamnose to each 7.5 mL dilution to induce catalase plasmid expression.
7. Transferred 1mL of each culture (including 6+9 and 8+9) to corresponding remaining test tubes.
8. Incubated at 37 degrees on a shaking incubator for 3 hours

1. Added each culture incubated with hydrogen peroxide to a 96-well plate.
2. Measured absorbance at 600 nm using Infinite M Plex plate reader.
3. Measured fluorescence with excitation at 570 nm and emission at 610 nm.

CRISPRi engineering

Plasmid Construction

1. Dilute primers to 100 uM using H2O.
   • Katg gRNA forward strand
   • Katg gRNA reverse strand
   • mScarlet3 gRNA forward strand
   • mScarlet3 gRNA reverse strand
2. Add 2uL of each partner strand to 14 uL of H2O and 2 uL of 10x annealing buffer
3. Anneal the strands: 5 minutes at 94 °C, then 70 x -1 °C per minute until the reaction vessel reaches 24°C

1. Dilute the annealed samples by 100x
2. Add 1 uL Ligase Buffer, 7 uL H2O, 0.5 uL diluted annealed sample, 0.5 uL pdCas9, 0.5 uL Bsa1 and 0.5 uL ligase to give sixfold excess of guide RNA
3. Cycle the strands: 50 x 5 minutes at 37 °C, 5 minutes at 16 °C, then 10 minutes at 37 °C, followed by 20 minutes at 80 °C

1. (strain) E. coli were transformed with the plasmids via electrocution. The cells were shocked in the presence of the plasmid, then diluted into LB to allow them to recover.
2. Selection plates were made with LB agar and chloramphenicol
3. The transformed cells were pelleted and plated onto the selection plates.
4. Plates were incubated overnight at 37 °C
5. Colonies were selected and grown overnight in LB broth with chloramphenicol
6. Plasmids were isolated by Isolate II Plasmid Mini Kit
7. DNA concertation was read by NanoDrop
8. The identity of the plasmids was confirmed via an enzyme digest, and sanger sequencing

1. (strain) E. coli were transformed with the following plasmid combinations as per the procedure in step 7
   • Combination 1 – Purified Katg pdCas9 Plasmid
   • Combination 2 – Purified mScarlet3 pdCas9 Plasmid + pET16 Rhamnose mScarlet3
   • Combination 3 – Purified mScarlet3 pdCas9 Plasmid + pCDF J23100 (constitutive) mScarlet3
2. Selection plates were made with chloramphenicol (to screen for combo 1), chloramphenicol + carbenicillin (to screen for Combo 2), and chloramphenicol + spectinomycin (to screen for combo 3)
3. The cells were pelleted and plated onto their respective plates.
4. 3-4 Colonies were selected from each plate and grown overnight in LB broth with their corresponding selection antibiotics.

CRISPRi Functionality Testing

To validate the function of the CRISPRi system, we replicated an experiment performed by Bradley (2021).

1. Combination 2 and 3 bacteria (from infusion transformation step) were used.
2. An example set up is shown below:

Where + indicates the addition of an inducer.

1. Each combination was grown in 3 mL lysogeny broth (LB)
2. Chloramphenicol stock of 33 mg/mL was added to LB to result in a final concentration of 33 µg/mL
3. Anhydrotetracycline stock of 100 µg/mL was added to the required conditions in table x. to result in a final concentration of 100 ng/mL
4. Rhamnose stock of 20% (m/v) was added to the required conditions in table above. to result in a final concentration of 0.2% (m/v)
5. Arabinose was added to result in a final concentration of 1.2 mM
6. A “high arabinose” concentration was also used of 26.6 mM (as a separate condition)
7. Following this, cultures were grown overnight at 37°C
8. On the next day, take 150 µL of overnight cultures and place into clear 96 well plate (Costar 96 U Bottom Transparent Polystyrene).
9. Absorbance at 600nm (OD600) was measured using Infinite M Plex plate reader.
10. 100 µL of the solution from the clear 96 well plate is transferred to a black 96 well plate (Costar 96 Flat Bottom Black Polystyrene).
11. mScarlet 3 fluorescence was measured using an excitation wavelength of 560nm and an emission wavelength of 620nm.

1. 3-4 colonies were selected from each of the purified pdCas9 plates grown overnight in selective LB broth.
2. 600 uL of incubated sample was added to 60 mL of new LB broth with 300 uL L-arabinose
3. The samples were incubated for 2.5 hours, then split into two. One replicate of each plasmid was given 30 uL aTc, and optical density at 600nm (OD600) was measured:
4. OD600 was measured at 30 minute intervals for 1 hour.
5. 0.1mM H2O2 was added to the samples
6. OD600 was recorded after 30 minutes of growth

Return to top