Abbreviation List

Table 1: Abbreviation List

Abbreviation	Full name
aTc	Anhydrotetracycline
AmpR	Ampicillin Resistance
CFU	Colony-Forming Unit
ddH2O	Double-Distilled Water
E. coli	Escherichia coli
KanR	Kanamycin Resistance
LB	Luria-Bertani
OD	Optical Density
PFA	Paraformaldehyde
PBS	Phosphate Buffered Saline
PDL	Poly-D-Lysine
SDS	Sodium Dodecyl Sulfate
TES	Tris Ethanesulfonic Acid, C6H15NO6S

Plasmid Transformation

Plasmid Transformation

1. Thaw the competent E. coli cells stored at -80°C on ice for 5 minutes.
2. Add 30 μL of competent E. coli cells to each tube, followed by the addition of 1 μL of plasmid DNA (100 ng/μL). Incubate on ice for 30 minutes.
3. Heat shock the mixture at 42°C for 45 seconds.
4. Immediately place the tube back on the ice for 2 minute and then add 1 mL of antibiotic-free SOC medium to the mixture.
5. Place the tube on a shaking incubator at 37°C and 220 rpm for 1 h.

Plasmid Miniprep

Plasmid Miniprep

We conducted this experiment using the same methods as those described in the 2022 Experiment (opens new window)

Plating

Plating

1. Take the tube from the shaking incubator, and centrifuge at 4000 rpm for 2 minutes.
2. Carefully remove the supernatant, leaving 100 μL, and resuspend the pellet by gently tapping the tube. Transfer the resuspended cells onto an agar plate.
3. Use a spreader to evenly distribute the cells on the agar plate.

SDS-PAGE

SDS-PAGE

Preparation of bacterial cell lysate for SDS-PAGE

1. Grow the cells overnight, to an OD₆₀₀ above 1.
2. Take 1 ml of OD₆₀₀ = 1 from each sample. Correct the sample volume to obtain an equivalent-size pellet.
3. Spin down the bacterial cells for 1 min at 13,000 rpm in a microfuge.
4. Aspirate any trace of supernatant with a vacuum line avoiding touching the pellet, but removing all liquid.
5. To each cell pellet, add 250 µL of distilled water, and resuspend the pellet. Add 250 µL 2x SDS sample buffer. Heat with a 95℃ heating block for 10 minutes. Store the samples at a -20℃ freezer until SDS-PAGE.

SDS-PAGE

1. Prepare the protein sample, as described above.
2. Tanon PrecastGel 4-20% was used.
3. Load the gel: Assemble the gel running stand. Fill the stand with 1 x MOPS running buffer and remove the combs from the gel. If needed, load 3 μL protein marker into the wells.
4. Electrophoresis: Cover the lid of the electrophoresis device, and start electrophoresis at 170 V until the dye front is nearly at the bottom of the gel.
5. Stain the gel: Submerge the whole piece of the disassembled gel with 0.1% Coomassie Blue dye for at least 30 min.
6. Destain the gel: Pour out the 0.1% Coomassie Blue dye and wash it using ddH₂O. Destain with a destaining solution for 30 min. Change the destaining solution several times to destain until it is clear.
7. Scan the gel.

IPTG induction experiment

IPTG induction experiment

We conducted this experiment as described in 2022 Experiments (opens new window)

Colony PCR

Colony PCR

We conducted this experiment as described in 2022 Experiments (opens new window)

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis

We conducted this experiment as described in 2022 Experiments (opens new window)

Algae Transfer

Algae Transfer

Step 1: Receiving and Initial Culturing

1. Upon receiving the algal strain, ensure thorough mixing of the algal culture in the test tube.
2. Under aseptic conditions, transfer the algal culture directly into a glass triangular flask with a volume ranging from 25 ml to 50 ml. Seal the flask tightly.
3. Place the sealed flask in a growth chamber with low-intensity lighting for 2-3 days.

Step 2: Controlling Cultivation Conditions

1. Maintain the cultivation temperature at 25°C.
2. Provide appropriate lighting conditions with an intensity of 1000-2000 lux.
3. Set a 12-hour light/12-hour dark cycle.

Step 3: Preparation and Subculturing

1. Prepare the corresponding growth medium.

2. Take 5-10 ml of the algal culture and add it to 10-20 ml of fresh growth medium.

3. Perform cultivation in a sterile triangular flask for 20-30 days (for strains with longer growth cycles, extend the cultivation time accordingly).

4. When the algal strain exhibits robust growth and a noticeable increase in biomass, perform a subculture. The subculture ratio for the next transfer should be 1:5 (algal culture to growth medium).

UV protocol

UV protocol

1. Preliminary Culture: Following plasmid transformation, incubate the E. coli SOC culturing in a shaking incubator at 37°C and 220 rpm for 2 hours.
2. Plating: After incubation, resuspend the cultured cells thoroughly and transfer 200 μL of the cell suspension onto a sterile agar plate. Use a sterile spreader to evenly distribute the cells across the agar surface. Wait until all lipid absorb.
3. UV irradiation: Cover half of the agar plate with a black paperboard to shield it from UV light, and expose the uncovered half of the plate to UV irradiation (power 6W) with combined wavelength of 254 nm and 365 nm for a total duration of 10 seconds
4. Post-UV Culture and Colony Counting: Incubate the plate inverted in a 37℃ incubator for 16 to 20 hours and count CFU on the plate.

Sucrose Utilizing Assay

Sucrose Utilizing Assay

1. Preparation of M9 minimal liquid medium: dissolve M9 solid dry powder (11.3 g/L) in ddH₂O to prepare M9 minimal liquid and autoclave at 121℃ for 15 minutes (Note: add filtered sucrose solution after autoclaving, otherwise the sucrose will be hydrolyzed into glucose and fructose!)
2. Medium Culture: the transformed bacteria (J101/J107/J109) were grown in LB medium in the presence of appropriate antibiotics at 37 ℃ overnight, diluting the initial LB culture of E. coli 1:100 with M9 medium with added sucrose (10g/L) or glucose (4g/L) at 37℃, 200 rpm
3. Measurement of growth curve: take 1 mL of bacterial culture at 0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 38 h, determine the concentration of the bacterial culture at 600 nm wavelength (OD₆₀₀ value), and plot the growth curve.
4. Provide extra factors: separate bacterial culture and add sucrose/glucose/MgSO₄/CaCl₂ respectively at 25 h

E. coli Aggregation assay

E. coli Aggregation assay

Table 2. Mixed Samples Table for Aggregation Assay

Strain	Name	Experimental group	Control Group 1	Control Group 2
DH5α-INPNC Ag3	Culture 1	Culture 1 + Culture 4		plain bacteria
DH5α-intimin Ag3	Culture 2		Culture 2 + Culture 4
DH5α-INPNC Nb3	Culture 3	Culture 2 + Culture 3
DH5α-intimin Nb3	Culture 4		Culture 2 + Culture 4

1. Cultures were incubated overnight in 7 mL Kanamycin LB medium (20-mL tube) at 37℃.
2. Then add 100ng/mL aTc, and 37°C 300 rpm incubated for 12 hours to ensure stationary phase and consistent final density across samples.
3. Following induction, using LB KanR medium to standardize the OD₆₀₀ to 1 across all the tubes. cultures were briefly vortexed and Cultures were mixed as described in the preceding table in a 1:1 ratio (each strain 600 μL per tube, and a 1.2 mL negative control group for plain bacteria, independent experiment repeat 3 times) at room temperature
4. Sampling was performed at 0, 3, and 6 hours by collecting 100 μL aliquots from the upper 25% of the mixture in each tube and transferred these samples into an EP tube, and stored in a refrigerator at 4 ℃.
5. After the final sampling, samples were resuspended and transferred to a 96-well assay plate, and the OD₆₀₀ was measured.

Cyanobacteria-E. coli Aggregation Assay

Cyanobacteria-E. coli Aggregation Assay

1. FACHB 410 was incubated for 48h in 4 mL CoBG-11 medium (20-mL tube) at 30℃. DH5α strains were also incubated for 48h in 2 mL CoBG-11 medium with 1 g/L glucose at 37℃.
2. Cultures were briefly vortexed , using the CoBG-11 medium to standardize the OD₆₀₀ and OD₆₈₅ across all the tubes.
3. Then Cultures were mixed in a 1:1 ratio (each 600 μL per tube (600 μL cyanobacteria and 600 μL E. coli induced/not induced with aTc ), independent experiment repeat 3 times) at room temperature
4. Sampling was performed at 0, 2, 6, and 24 hours by collecting 100 μL aliquots from the upper 25% of the mixture in each tube. Transfer these samples into an EP tube and store them in a refrigerator at 4 ℃.
5. After the final sampling, samples were resuspended and transferred to a 96-well assay plate, and the OD₆₀₀ and OD₆₈₅ were measured.

Medium (named CoBG-11)

CoBG-11 was designed based on BG-11 medium and optimized for E. coli growth by supplementing with 150 mM NaCl, 4 mM NH₄Cl, and 3 g/L TES. NaCl and NH₄Cl were used to maintain the cell survival of E. coli.

Antifreeze Assay

Antifreeze Assay

1. Cultures were incubated overnight at 37℃ 300 rpm in 2 mL LB liquid median.
2. After reaching an OD₆₀₀ value of 1, subject the culture to a cold treatment at 0°C(Cold treatment condition: Place the culture tubes in an ice-water bath and store them in a 4°C refrigerator. Change the ice every 12 hours. )
3. Samples for CFU counting are taken at 0, 12, 24, 48, 60, 72, 84, and 96 hours during cold-treated cultivation. Independent experiment was repeated 3 times.

Desiccation Survival Assay

Desiccation Survival Assay

1. Measure and record the dry weight of each EP tube. Adjust the OD₆₀₀ values of the experimental group and the control group to 1.
2. For each sample， dispense 1 mL bacterial solution into two separate EP tubes. Centrifuge at 6000 rpm for 5 minutes.
3. Carefully remove the supernatant before desiccation in a speedvac concentrator for 6.5 hours. Then measure the dry weight of each bacterial pellet. Place the EP tubes containing bacteria on an analytical balance and record their weight. Bacterial dry weight is calculated as the recorded weight minus the dry weight of the empty EP tube measured in step 1.
4. Re-suspend each EP tube pellet in 1 mL of LB medium without antibiotics. Dilute it 10⁵ times for CFU counting.

CFU Counting

CFU Counting

Table 3: Dilution procedure for CFU counting

Dilution ratio	Solution Proportions
The First Diluted Tube	100 μL Experimental Groups Bacterial Solution (OD600 1 = 108/mL) + 9.9 mL ddH2O
The second Diluted Tube	100 μL First Diluted Solution + 9.9 mL ddH2O
The third Diluted Tube	1.0 mL Second Diluted Solution + 9.0 mL ddH2O
Final Dilution ratio	1 : 100,000

1. Dilute the bacterial culture sample (100 μL) to 1:100,000 and take 100 μL from the third diluted tube to perform plate culturing.
2. After incubating at 37°C for 16 hours, for each sample, CFU count = the number of colonies on the agar plate × 10⁵.

Fluorescence Microplate Read

Fluorescence Microplate Read

1. Take 500 μL bacterial culture and add 500 μL PBS. Centrifuge at 6000 rpm for 3 minutes, then discard the supernatant.
2. Resuspend the pellet in 100 μL PBS, then add 1 mL 4% PFA in PBS to fix the sample for 20 minutes.
3. Centrifuge at 6000 rpm for 3 minutes, discard the supernatant, and resuspend the pellet in 500 μL PBS.
4. Dilute the bacterial liquid with PBS to achieve dilution factors of 0.5, 0.25, and 0.125. Take 100 μL from each dilution factor, as well as 100 μL undiluted bacterial liquid and 100 μL PBS.
5. Place these samples into a microplate reader (BioTek Cytation 3) and measure the fluorescence intensity of GFP, RFP, and the OD₆₀₀ value.
6. Adjust the gain coefficients based on the initial measurements taken during the first reading. For GFP and RFP, gain coefficients should be consistent.
7. Calculate the RFP/GFP ratio of fluorescence intensity.

For RBS or stem-loop testing, the following was used

	Experimental Group	Control Group 1	Control Group 2
Plasmid Design	T7 promoter + testing RBS + stayGold + testing stem-loop + Twister ribozyme + target RBS + mScarlet + T7 terminater	T7 promoter+T7 RBS + stayGold + stem-loop + Twister ribozyme + T7 RBS + mScarlet + T7 terminater	Plain E. coli

Biofilm Growth Imaging

Biofilm Growth Imaging

1. E. coli cultures expressing intimin-Ag3 and intimin-Nb3 were incubated overnight at 37°C，300 rpm in 2 mL of LB KanR medium.
2. The cultures were grown to the exponential phase (OD₆₀₀ = 0.5-0.6) for subsequent steps.
3. Chambered glass-bottom dishes were briefly treated with 0.1% PDL for 10 seconds.
4. The two strains of E. coli were mixed by forcely pepetting, and applied to the glass.
5. After several washes with LB KanR medium, 500 μL of LB KanR medium was added. The low initial inoculation density enabled isolated biofilm clusters to form.
6. Upon identifying the locations of the founder cells, imaging was initiated on a motorized microscope at 37°C, with a 150x oil objective lens.

EPS Adhesion Assay

EPS Adhesion Assay

1. The E. coli cultures expressing EPS were incubated overnight at 37°C, 300 rpm in 2 mL of LB KanR medium until the OD₆₀₀ reached 1.
2. We followed the PDL "in-chamber" method, and chamber setup described in reference ^[1] to perform chamber-based microscopy.

References

1. Wang, Y.-K., Krasnopeeva, E., Lin, S.-Y., Bai, F., Pilizota, T., & Lo, C.-J. (2019). Comparison of Escherichia coli surface attachment methods for single-cell microscopy. Scientific Reports, 9(1), 19418. https://doi.org/10.1038/s41598-019-55798-0 ↩︎