Abbreviation List

Table 1: Abbreviation List

Abbreviation	Full name
TEM	Transmission Electron Microscope
Ni2+	divalent nickel ion
ddH2O	Double-Distilled Water
E. coli	Escherichia coli
MOIs	multiplicity of infections
NGM	Nematode Growth Medium
LB	Luria-Bertani
OD	Optical Density
PBS	Phosphate Buffered Saline
SDS	Sodium Dodecyl Sulfate

Ni²⁺ Concentration Measurement

Prepare the Ni²⁺ Standard Curve

I. Principle

1. In an ammonium citrate-ammonia water medium, iodine acts as an oxidizing agent and promotes the reaction between nickel ions and dimethylglyoxime, which form a red wine-colored complex.
2. The formed complex has a maximum absorption at the wavelength of 530 nm, thus a spectrophotometer can be used to measure its absorbance.
3. By preparing the Ni²⁺ standard curve, the concentration of nickel in the sample can be quantitatively determined.

II. Reagents

• 500 g/L Tribasic ammonium citrate solution
• 0.05 mol/L Iodine solution
• 5 g/L Dimethylglyoxime
• 50 g/L Na₂EDTA
• 10 mg/L Nickel standard stock solution: Weigh 0.20325 g nickel chloride hexahydrate (NiCl₂·6H₂O), dissolve in 5 mL ddH₂O.

III. Experimental Procedures

1. Prepare the following components in EP tubes (2 mL volumes, add from left to right):

Group No.	[NiCl2] mg/L	ddH2O / μL	Nickel Standard Stock / μL	Ammonium Citrate / μL	Iodine / μL	Dimethylglyoxime / μL	Na-EDTA / μL
1	0	1440	0	160	80	160	160
2	0.5	1340	100	160	80	160	160
3	1	1240	200	160	80	160	160
4	1.5	1140	300	160	80	160	160
5	2	1040	400	160	80	160	160
6	3	840	600	160	80	160	160
7	4	640	800	160	80	160	160

For each EP tube, shake well after adding reagents and set aside for 5 minutes. Shake before measuring each tube. Measure its absorbance at wavelength 530 nm. Use group 1 as a blank control, and plot the curve by subtracting the absorbance of group 1 from groups 2-7. Rinse the cuvette thoroughly with ddH₂0 between measurments.

2. Plot the absorbance and nickel ion concentration (blue dots). Use line regression to obtain the Formual 1.
3. Three sets of divalent nickel solutions with known concentrations are prepared to verify the accuracy of our standard curve. For the test group, prepare the following components in EP tubes (2 mL volumes, add from left to right):

Group No.	[NiCl2] mg/L	ddH2O	Nickel Standard Stock / μL	Ammonium Citrate / μL	Iodine / μL	Dimethylglyoxime / μL	Na-EDTA / μL
1	0	1440	0	160	80	160	160
2	1.25	1190	250	160	80	160	160
3	2.5	940	500	160	80	160	160
4	3.5	740	700	160	80	160	160

IV. Results

Figure 1: Standard Curve of Abs~530~ and NiCl~2~ Concentration

Formual 1:  Abs = 0.04343*C + 0.7438 [R Square = 0.9984]

Measurement of Samples

1. Pellet the bacteria by 8000 rpm centrifugation for 5 minutes. Use the supernatants for measurements. Prepare the following components in EP tubes (2 mL volumes, add from left to right):

Group No.	Culture Medium Initial [NiCl2] mg/L	ddH2O	Bacterial Supernatant / μL	Ammonium Citrate / μL	Iodine / μL	Dimethylglyoxime / μL	Na-EDTA / μL
1	20	1190	250	160	80	160	160
2	30	1190	250	160	80	160	160
3	50	1240	200	160	80	160	160
4	100	1390	50	160	80	160	160
5	150	1390	50	160	80	160	160

For each EP tube, shake well after adding reagents and set aside for 5 minutes. Shake before measuring each tube and rinse the cuvette. Then measure its absorbance at wavelength 530 nm. Use the color-developing solution without NiCl₂ as a blank control.

Phage Resistance Assay

1. Cultivate the bacteria for approximately 4 hours to reach the logarithmic growth phase, adjusting the OD₆₀₀ to 0.5.
2. Take 1 mL of bacterial culture and add phages to achieve MOIs of 0.5, 1, and 2 (E. coli concentration is estimated to be 1 x 10⁸ at OD₆₀₀ = 1; and the phage concentration is 2 x 10¹⁰/mL, diluted from a 2 x 10¹³ stock).
3. Incubate at 37°C for 30 minutes while pre-warming the agar plates.
4. Take 150 µL of the bacteria/phage mixture and spread it onto the plate.
5. Incubate overnight at 37°C.

The phage carries Kan resistance. If the bacteria was infected by the phage, colonies form on a Kan plate.

Ecotoxicity Testing

Reagents

• NGM Preparation

  1. Add 2.5 g peptone, 3 g NaCl, 20 g agar, 25 mL of 1 mol/L phosphate buffer (108.3 g KH₂PO₄, 35.6 g K₂HPO₄, pH 6.0, made up to 1 L), 1 mL of 1 mol/L CaCl₂ solution, 1 mL of 1 mol/L MgSO₄ solution, dissolve in 1 L ddH₂O.
  2. Sterilize at 121°C under high pressure for 20 minutes.
  3. Cool down to approximately 55°C at room temperature, then add 1 mL of a 5 mg/mL cholesterol solution (prepared in anhydrous ethanol).

• M9 Buffer Preparation: Add 3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL of 1 mol/L MgSO₄, dissolve in 1 L ddH₂O.

• Nematode Lysis Solution (prepare before use): Add 1 mL of 5 mol/L NaOH, 1 mL of 10% NaCl, dissolve in 7 mL ddH₂O.

Experimental Procedures

Synchronization of Nematodes:

1. Rinse the nematodes with 2 mL ddH₂O, transfer them to a centrifuge tube, and centrifuge at 3000 rpm for one minute.
2. Discard the supernatant carefully. Add 1 mL of nematode lysis solution, vortex thoroughly for 10 minutes.
3. Centrifuge at 5000 rpm for one minute, discard the supernatant.
4. Add 1 mL of M9 buffer, vortex thoroughly, centrifuge at 3000 rpm for one minute. Discard the supernatant. Repeat 2-3-4 process three times, leaving approximately 100 µL of M9 remains in the centrifuge tube after the final round.
5. Dispense the solution on NGM medium (coated with E. coli OP50 lawm) and culture at 20°C. Collect L2-stage nematodes after approximately 20 hours.

Locomotion Measurement:

1. Collect the L2-stage nematodes and deposit them on the medium.
2. Incubate at 20°C for 18 hours and capture their locomotion using a steroscope.
3. Analyze the recorded movement trajectories utilizing ImageJ Plugins Animal Tracker.

Other experimental methods

• TEM sample preparation and imaging was carried by the EM Core Facility of SKLGE.
• Fluorescence imaging of BBa_K5115057 was carried out on a motorized spinning-disk confocal microscope at room temperature, with a 150x oil objective lens (NA 1.45). Agarose pad was prepared as described previously.
• Cyanobacteria-E. coli Aggregation Assay was carried out as described previously.
• IPTG induction was carried out as described previously.
• Plasmid miniprep was conducted as described previously.
• DNA agarose gel electrophoresis was carried out as described previously.
• Overlap extension PCR was used to generate fusion proteins.
• PCR was used to generate inserts and vectors for ligase-free cloning.
• Restriction enzyme digestion was used to generate fragments from ribozyme connected parts, specifically PmeI to generate inserts and SwaI for vectors.
• Inserts and vectors were ligated using ClonExpress reaction.
• Bacteria transformation and plating were conducted as described previously.
• Bacteria colony PCR with the listed primers was used to screen plasmids before sending to Sanger sequencing.
• Bacteria cryopreservation and recovery were performed as described previously.