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1. PCR Reactions
SoxRS regulon Amplification

The PCR was carried out using the primers PPC1 and PPC2 and Q5 polymerase (New England Biolabs) following a protocol of 20 seconds of initial denaturation at 98 ºC, 30 cycles of 98 °C for 10 seconds, 59.5 °C for 30 seconds, and 72 °C for 30 seconds, and a final extension at 72 °C for 5 minutes.

2. GFP Amplification.

The PCR was carried out using the primers PPC3 and PPC4 and Q5 polymerase (New England Biolabs) following a protocol of 20 seconds of initial denaturation at 98 ºC, 30 cycles of 98 °C for 10 seconds, 61.5 °C for 30 seconds, and 72 °C for 30 seconds, and a final extension at 72 °C for 5 minutes.

3. Gel Electrophoresis

Both PCR product were run on a 1% agarose gel and extracted using the New England Biolabs gel extraction kit using the recommended protocol.

4. Golden Gate Assembly

The two amplicons were cloned into the pBR322 vector by adding 75ng of pBR322 and 30.1ng of SoxRS regulon and 23.4ng of GFP amplicon to the golden gate assembly mix (NEB, E1600S). Following the NEB protocol the mixture was incubated at 37°C for 1 hr and at 55°C, 5 mins.

5. Transformation

The PixCell construct was transformed into DJ901 and Turbo cells using the KCM heat shock protocol.

6. Addition of the Degradation Tag

Turbo cells containing the PixCell construct were grown overnight and miniprepped following the NEB protocol in order to obtain template DNA for the following PCR reaction. The PCR was carried out using the primers PPC5 and PPC6 and Phusion polymerase (New England Biolabs) with 3% DMSO. Following a protocol of 30 seconds of initial denaturation at 98 ºC, 30 cycles of 98 °C for 10 seconds, 65 °C for 30 seconds, and 72 °C for 3 minutes, and a final extension at 72 °C for 10 minutes. These primers produce complementary overhangs which when ligated together add a degradation tag we designed to the 3’ end of the coding region of GFP.

7. Ligation

The ends of the PCR amplicon produced from the primers PPC5 and PPC6 were ligated using T4 DNA ligase (NEB) following the recommended NEB protocol.

8. Transformation

9. The PixCell construct with deg tag was transformed into DJ901 cells using the KCM heat shock protocol.

• Autoclaved LB media
• Autoclaved 10x concentated LB media
• 500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)
• 1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)
• 500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )
• 2% Sodium Sulfite Solution
• Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).
• 37°C shaking incubator
• BMG Labtech FLUOstar plate reader
• 4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)
• 14 mL culture tubes
• autoclaved eppendorfs

Work in very sterile conditions. To prevent contaminations in the blanks, use triple antibiotics.
Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use.
We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water. Mastemixes have double the working concentrations, as 100 uL of solution is then diluted with 100 uL of liquid cultures.

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1. Grow starter liquid cultures of cells (Day 0, 10-15 min preparation + overnight)

Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below:

Label	E. coli strain	Construct	Antibiotic (Ab)	V Ab (uL)	V LB (mL)
PC1	DJ901	BBa_K2862021	Kan + Amp	5 + 5	5
PC2	DJ901	BBa_K2862022	Kan + Amp	5 + 5	5
-	DJ901	none	Kan	5	5
+	DJ901	GFP_Boo	Kan + Chl	5 + 5	5
Blanks	None	None	Kan + Amp + Chl	5 + 5	5

With a P20 pipette tip we picked the desired colonies from an agar plate and released the contaminated tip in the culture tip (for PC1, PC2 and Pos the picked colonies appeared fluorescence under a UV transilluminator). We then place inoculated culture tubes in a 37° C in a shaking incubator overnight. For faster growth the angle between the vertical axis of the tube and the shaking plane of the incubator was set at 45°)

2. Prepare mastermixes (30 min)

Mastermixes for each redox molecule concentration prepared in 1.5 mL eppendorfs. The mastermixes are enough to fill 2 96-well microplates with 100 uL of solutions in each well, in order to have two replicate of the same experiment. Take 8 autoclaved 1.5 mL eppendorfs and prepare them as described in the table below.

Mastermizes for Day 1 and Day 2 :

Condition	V Pyo (uL)	Kan (uL)	LB 10x uL	Autoclaved Water (uL)	Tot Vol (uL)
A = 0 uM Pyo	0	2.44	122	1098	1220
B = 0.01 uM Pyo	0.0976	2.44	122	1097.9024	1220
C = 0.025 uM Pyo	0.244	2.44	122	1097.756	1220
D = 0.05 uM Pyo	0.488	2.44	122	1097.512	1220
E = 0.1 uM Pyo	0.976	2.44	122	1097.024	1220
F = 0.25 uM Pyo	2.44	2.44	122	1095.56	1220
G = 0.5 uM Pyo	4.88	2.44	122	1093.12	1220
H = 1 uM Pyo	9.76	2.44	122	1088.24	1220

Mastermixes for Day 3 and Day 4:

Condition	V Pyo (uL)	Kan (uL)	LB 10x uL	Autoclaved Water (uL)	Tot Vol (uL)
A = 2.5 uM Pyo	24.4	2.44	122	1073.6	1220
B = 5 uM Pyo	48.8	2.44	122	1049.2	1220
C = 10 uM Pyo	97.6	2.44	122	1000.4	1220
D = 25 uM Pyo	244	2.44	122	854	1220
E = 50 uM Pyo	488	2.44	122	610	1220
F = 100 uM Pyo	976	2.44	122	122	1220

We transferred master mixes into clean eppendorf tubes, with volumes specified below, to add the right antibiotics:

Condition	Volume of A-H Mastermix (uL)	Ampicillin (uL)	Chloramphenicol (uL)
PC1 + PC2	406	2.44	0
+	203	0	2.44
-	203	0	0
Blanks	406	2.44	2.44

This gave a total of 32 eppendorf tubes for Day 1 and 24 on Day 2, ready to be transferred into 96-well plates.
3. Fill 96-well plate with media solutions

Add 100 uL of media in the desired wells, according following the label on mastermixes and table below:

	1	2	3	4	5	6	7	8	9	10	11	12
A	PC1	PC1	BA	PC2	PC2	BA	-	-	BA	+	+	BA
B	PC1	PC1	BB	PC2	PC2	BB	-	-	BB	+	+	BB
C	PC1	PC1	BC	PC2	PC2	BC	-	-	BC	+	+	BC
D	PC1	PC1	BD	PC2	PC2	BD	-	-	BD	+	+	BD
E	PC1	PC1	BE	PC2	PC2	BE	-	-	BE	+	+	BE
F	PC1	PC1	BF	PC2	PC2	BF	-	-	BF	+	+	BF
G	PC1	PC1	BG	PC2	PC2	BG	-	-	BG	+	+	BG
H	PC1	PC1	BH	PC2	PC2	BH	-	-	BH	+	+	BH

4. OD600 matching

After overnight growth, we diluted the all the cultures to an OD of 0.1. We diluted using a FLUOstar plate reader measuring single endpoint absorbance at 600 nm. We added 100 uL of LB + antibiotic into a blank well. We filled the the microplane with the cell culture solutions and took endpoint measurements of the filled wells and blanked them with the blank well. We note down the OD600 value of the wells and dilute them to tan OD600 of 0.1 according to the cell dilution calculator formula described below: (a value of 0.1 is suggested for a volume of 100 uL

Cell dilution calculator: Volume of LB+Antibiotic to add in culture tube = [( Vcells * ODcell)/desired OD] - Vcells E.g. to dilute 5mL of cells at an OD600 of 0.3 to OD of 0.1 = [ (5mL*0.3)/0.1 - 5] =10 mL

5. Add cells to 96-well plate

We pipetted 100 uL of liquid cultures ot OD600= 0.1 into the corresponding wells,following 96-well microplate layout and culture tubes label.

6. Set up the script in the plate reader and start experiment

For Pyocyanin experiments:

We set up a script on the BMG Labtech Omega control software to perform endpoint absorbance measurements at 600 nm and fluorescent intensity measurements every 10 minutes for 24 hours (288 cycles for 600 seconds cycles time). Both absorbance and fluorescence measurements were recorded from the bottom of the plate. The excitation/emission wavelengths for GFP detection was set up at 475-510 nm. The plate reader was set up to shake at a double orbital mode at an RPM of 200.

7. Data analysis

We exported the raw data excel spreadsheet and imported into MATLAB software. We calculated average OD600, standard deviation and standard errors (STDEV/SQRT(4), where 4 was our number of replicates. We performed the same calculations for fluorescence values. We divided fluorescence values at each time point by the corresponding OD600 values, to normalise GFP expression by the number of cells. We calculated standard deviations and standard errors analogously for OD600. We then plotted OD600 as a function of time, GFP/OD600. From these plots we identified the time at which cell growth and GFP expression reached a steady state and called this time T_survive. We then took the GFP/OD600 values at T_survive for each construct and plot them against pyocyanin concentration to plot concentration curves, as shown in the “Results” section. Similar analysis was performed for ferrocyanide and ferricyanide and PMS experiments

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Square-wave voltammetry was obtained with the setup descripted in the results section. (LINK TO RESULTS). Solutions in the flask containing the reference and working electrode were identical to the solutions in the respective flasks containing the counter electrode. Square-wave voltammetry was performed with the PalmSens 4-channel MultiEmStat3 between -0.3 and +0.5 Volt. The counter electrodes contained 60% Carbon with a diameter of 5mm. Working electrodes contained 90% Carbon with a 2mm diameter. Salt bridges were consists of 3% agar containing 1 molar potassium chloride inside plastic tubing. The reference electrode was produced by placing a silver wire within 3% agar containing 3 molar potassium chloride inside plastic tubing. Square wave voltammetry was performed with the following treatment settings: 10s equilibrium time, E step 0.005V, amplitude 0.05V, frequency 25 Hz, e beginn and e end -0.3 to 0.5. Amperometry was performed in a plate containing the final conditions: 10mM FCN(R), 2.5 uM Pyocyanin, 0.02% Sulfite, Ampicillin, Kanamycin and cells containing the pixCell construct with the following treatment settings: 10s equilibrium time, time interval 0.05s run time up to 65000 seconds, V=+0.5.

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Three 5mm holes were drilled into 9cm agar plates. Working, counter reference electrodes were prepared as described here (LINK TO RESULTS SECTION OF Electrochemistry of Redox Modulators). Electrodes were inserted into the plate penetrating 2mm. Electrodes were then glued to the plate. Solutions containing 10mM FCN(R), 2.5 uM Pyocyanin, 0.03% Sulfite, Ampicillin and Kanamycin were pitpetted into a 50ml falcon tube. 50ml of molten 1% agar was added to the falcon tube. Shaking ensured that the solution was well mixed. After mixing 22ml of the solution was pipetted onto the agar plate. It was ensured that agar covered the electrodes fully. After the agar was set, 100uL of cells containing the PixCell construct Deg Tag at an OD of 0.3. were plated onto the agar. Afterwards the plate was turned upside down, closed and sealed using tape. The setup was then placed into a 37°C incubator. Potentials were then applied overnight for 13h as described here. (link results amperometry) To investigate fluorescence the agar was transferred onto a fresh plate without electrodes penetrating from the bottom. Fluorescence was measured using the Fujifilm FLA-5000 Fluorescent Image Analyser on the following settings: Laser wavelength: 250nm, filter: FITC, gradation: 65536 (16bit), resolution: 50uM. Images where then imported into imageJ for image analysis. Minimum brightness was set to 13867 in all images to discard small fluorescence values which correspond to background fluorescence.

All the experiments for this part of the project were performed on E. coli DH5α, origin strain for DJ901, as this last one could not be made super-competent (109 cfu / μg pUC19 DNA) enough to transform BASIC assembly products. Individual parts were integrated in chloramphenicol resistant high copy plasmid pSB1C3_BASIC vectors and assemblies were performed in ampicillin resistant mid copy plasmid p15A vectors. All individual parts were synthesised and ordered as gblocks with BASIC prefix and suffix through IDT. The linkers used were provided by Biolegio and the adaptors were courtesy of Dr. Baldwin’s lab at Imperial College London.

Parts assembly for library generation was performed as described in Storch, Casini et al., 2015. Briefly, linkers were prepared adding 49 µl of annealing buffer ( 10mM TRIS-HCl buffer pH7.9, 100mM NaCl, 10mM MgCl2 ) to 0.5 µl (1 µM) of linker and 0.5 µl (1 µM) of adapter oligos, and incubating at 95ºC for 1 min. Once linkers were ready they were mixed with the parts for the digestion/ligation reaction and incubated at 37ºC for 1 hour, 20ºC for 20 min, 65ºC for 20 min. Each 30 µl reaction had 3µl of ATP, 3µl of NEB4, 3µl BSA, 5µl of prefix linker (1µM), 5µl of suffix linker (1 µM), 1µl of BsaI (20 U/µl), 0.5 µl of T4 ligase (400 U/µl), 1µl DNA part (200 ng/µl) and water. Parts and linkers were mixed according to the following table.

We determined that PC_A1 is our base construct as it is Pixcell construct in library format. This means that it has the same transcription factor (SoxR-0) and the same promoter (pSoxS-0) only that this one in Pfam1 promoter architecture. Therefore, this construct should give us the most similar results to Pixcell construct and show the effect of library architecture.

Promoter induction assay

The best colonies from each succesful construct were characterised in an induction assay with pyocyanin. Assays were performed with different concentrations of pyocyanin (0-100 µM) and 100 µl of OD 0.1 cells in BMG Clariostat plate readers at 30º C.

Part growth assay

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