10 g/L tryptone
5 g/L yeast extract
5 g/L NaCl
15 g/L agar bacteriological agar type E
Dissolve in demineralized water, stir and autoclave.

Plasmid extraction (QIAGEN Kit)

1. Preparation in advance: Add LiseBlue Regent into P1 Buffer (1:1000), and then add RNase A solution. After mixing, store in the refrigerator at 4 ℃. Add ethanol (96~100%) to Buffer PE.
2. The bacterial solution in the shaking tube is poured into a 2 mL centrifuge tube and centrifuged at 13,000 rpm (QIAGEN centrifuge) for 5 min (pay attention to the balance) to obtain bacterial precipitation. After the supernatant is poured out, the remaining liquid is poured into the shaking tube, and the above steps are repeated until the liquid in the shaking tube is poured out (the waste liquid bucket containing the supernatant should be added with diluted 84 disinfectant, and it should be left in the ventilator for one day).
3. Pour off the supernatant after the last centrifugation, remove Buffer P1 from the refrigerator at 4 °C, and add 250 uL of Buffer P1 to the centrifuge tube. The mixture is blown repeatedly with a 200 uL pipette until the liquid in the centrifuge tube turned milky and cloudy, and there is no bacterial precipitation on the wall.
4. Add 250 uL of Buffer P2 to the centrifuge tube and invert the centrifuge tube for several times to mix the liquid until the liquid becomes clear and appears light blue.
5. Add 350 uL Buffer N3 and mix the tube upside down (gently) Immediately until the liquid in the centrifuge tube becomes colorless and white flocculent precipitate appears.
6. Centrifuge at 13,000 rpm for 10 min, during which follow-up work can be prepared.
7. Use a pipette gun set to 800 uL to absorb the supernatant after centrifugation, then add it to the adsorption column, and centrifuge it at 13,000 rpm for 1 min.
8. Pour out the filtered liquid from the adsorption column and add 750 uL Buffer PE (containing alcohol, the liquid is easy to drip when the pipette is drawn) to wash the adsorption column, and centrifuge it at 13,000 rpm for 1 min.
9. Discard the filtered liquid from the adsorption column. After that, place the adsorption column on a new 1.5 mL centrifuge tube, and centrifuge it at 13,000 rpm for two minutes.
10. Discard the filtered liquid from the adsorption column, place the adsorption column on a new 1.5 mL centrifuge tube for 1 minute until the alcohol volatilization, and add 50 uL Buffer EB to the adsorption column (note that it should be added to the adsorption column, the nozzle should be deep, and replace the nozzle if it hits the tube wall). It is allowed to stand for 1 min and centrifuged at 13,000 rpm for 1 min.
11. Discard the adsorption column and write the name of the plasmid, preparation time and the name of the preparator on the centrifuge tube, and determine the DNA concentration.
12. The plasmids are stored in the refrigerator at -20 ℃.

Enzyme digestion

Plasmid DNA: X μL (according to the actual concentration)
Restriction enzyme 1: 2 μL
Restriction enzyme 2: 2 μL
10×buffer: 5μL
ddH₂O: 50-X μL
The total reaction system is 50 μL, add samples in PCR tubes and mix well, incubate at 37 ℃ for 1-3 h, electrophoresis verification.

Polymerase Chain Reaction (PCR)

Overlapping PCR

Bacteria colony PCR

Monoclonal colonies (×12) are selected in 800 uL of a resistant LB system with antibiotics. The culture is placed in a constant temperature shaker for 2 h.

Gibson reconstruction

Reaction procedure: 50℃; 15min

For the single-fragment homologous recombination reaction:
Optimal amount of cloning vector =[0.02× number of cloning vector base pairs]ng(0.03 pmol);
Optimal amount of inserted fragments =[0.04× number of inserted base pairs]ng (0.06 pmol)

Gel electrophoresis

1.Determine to use small system (40 mL) or large system (60 mL).
2. Use a special conical flask with 1% agarose concentration. For example, if 43 mL of 1×TAS buffer is added, 0.43 g of agarose powder should be added.
3. Shake the flask and heat it in the microwave for 2 min until the agarose dissolves.
4. Take out the conical flask with heat insulation gloves and rinse it under cold water for a few seconds until it is not hot.
5. Use a pipette gun to add 1/10000 volume of dye (Gelred 10000×) and shake evenly.
6. Add the liquid in the conical flask to the glue-matching plate, and the excess liquid will flow out. Remove the air bubbles from the glue to the place that will not affect the glue-running, and let the gel cool for 20 min to solid state.
7. Place the solid gel in the electrophoresis tank (completely immersed in the liquid) and pay attention to the electrode direction.
8. The plasmid is removed from the refrigerator at -20 ℃, and 10 uL Purple Loading Dye is added to each tube.
9. Blow the mixed liquid evenly with a pipette gun, then absorb all the liquid and add it to the spot sample hole, and add 10 uL DNA marker to the leftmost spot sample hole.
10. Electrophoresis is carried out at 120 V voltage and suitable time according to the DNA size.

Gel recovery (Bioengineering Kit)

1. The target glue block is cut with a blade under the blue light glue cutter (the specificity of PCR product is confirmed by marker, and the glue is cut vertically in the shape of a well).
2. Preheat 200 uL of Elution Buffer at 55 °C.
3. Weigh the gel block and add Buffer B2 at the ratio of 300~ 600 uL/100 mg (directly add 600 uL Buffer B2 in this experiment).
4. Put the mixture at 50 ℃ for 10 min, then shake and mix once every 2-3 min until dissloved.
5. Adjust the pipette gun to 800 uL (Larger volume after the gel dissolved. If 800 uL is not enough, repeat this step) and transfer the mixed solution to the adsorption column. Next, centrifuge at 8,000 × g for 1 min, and discard liquid in the collection tube.
6. Add 300 uL Buffer B2 to the adsorption column, centrifuge at 9,000 × g for 1 min, and discard liquid in the collection tube.
7. Add 500 uL Wash Buffer (completed with absolute ethanol), centrifuge at 9,000 × g for 1 min. Repeat this step.
8. Put the empty adsorption column on the collection tube, centrifuge it at 9,000 × g for 1 min, and then let it stand for 1 min until the alcohol volatilizes.
9. Put the empty adsorption column on a new 1.5 mL tube. Add 30 uL preheated Elution Buffer to the center of the adsorption column, stand for 1 min, centrifuge 9,000 × g for 1 min, and discard the adsorption column.
10. Determine DNA concentration.
11. The obtained DNA solution is stored at -20 ℃.

The plasmid PET28A-FMO-ptUGT is transformed into strain DE3 and cultured overnight in LB medium with resistance. The next day, the bacterial solution is diluted at a fold of 100 and incubated at 37 °C on a shaker at 200 rpm until OD 600 reached 0.6. After that, 0.3 mM IPTG is added for induction overnight (the induced bacterial solution can be resuspended by buffer solution of HEPES with a concentration of 50 mM and pH 7.0, sodium chloride solution with a concentration of 300 mM, imidazole solution with a concentration of 25 mM and pH 8.0, and DTT solution with a concentration of mM.) Finally, UGT is purified by cleavage.

Reaction detection

After incubation overnight to saturation, the bacteria solution is diluted in 50 mL M9 medium until OD 600 reached 0.05. Then 26 mM Trp, 2% glycerol, 0.2% glucose, 1 mM IPTG are added and the reaction is performed at 37 °C and shaked at 200 rpm. During this time, the content of bacteria and reactants should be measured regularly.

Expression and reaction of TnaA-FMO

Single colonies are selected and incubated in LB medium with antibiotics overnight. The next day, the bacterial solution is diluted 1:100 and incubated in a 37 °C and 200 rpm shaker until the OD 600 value reached 0.6. After that, 0.3 mM IPTG is added for overnight induction. Next, the bacterial solution is centrifuged at 3,500 × g for 10 min, the excess LB is washed with PBS, and the cells are resuspended with NPB solution of 50 mM and pH=8.0. Final reaction system is: total volume 4 mL,OD 600=20,Trp (BR-TRP) 1 mM, glucose 0.6%. The reaction is performed at 30 °C for 24 h.

After the reaction, the bacterial solution is partitioned into a 1.5 mL centrifuge tube, centrifuged at 13,000 × g for 10 min, and indigo is dissolved with 50% DMSO in H2O or 100% DMSO.

Expression and reaction of Fre-SttH

Single colonies are selected and incubated overnight in LB medium with antibiotics. The next day, the bacterial solution is diluted 1:100 and incubated in a 37 °C and 200 rpm shaker until the OD 600 value reached 0.6. Then 0.3 mM IPTG is added for overnight induction. Next, the bacterial solution is centrifuged at 3,500 × g for 10 min, the excess LB is washed off with PBS, and the cells are resuspended with NPB solution with a concentration of 50 mM and pH=8.0. The final reaction system is as follows: The total volume is 4 mL, OD 600=20, Trp 2.5 mM, glucose 0.6%, NaBr 300 mM, 30 °C. Reaction for 24h.

Preparation of SDS-PAGE samples

The induced bacterial solution is centrifuged at 4,000 rpm for 10 min and resuspended in PBS. Then 15 uL of whole cell sample is taken. The remaining bacterial solution is lysed by ultrasonic wave and centrifuged at 13,000 × g for 2 min. Next, 15 uL of supernatant sample is taken. After mixing with sample buffer for protein, the sample is boiled at 100°C for 5min before loading.

Br-trp, Trp:
1 mL of reaction solution is centrifuged at 13,000 × g for 10 min, and the supernatant is filtered through a 0.22 um filter.

Indole, Br-indole:
200 uL of reaction solution is added with 400 uL CHCl3 and removed 200 uL CHCl3 after vortex. The mixture is evaporated in a fume hood and the remainder dissolved in 100 uL of ethyl acetate.

Indican:
The reaction solution is filtered through a 0.22 um filter and diluted with 10 times water.

LC-MS sample processing

Samples to be tested: tryptophan, 6-chloro-tryptophan, 6-bromotryptophan
Chromatographic Column: C18 chromatographic column
Mobile phase:
A: 0.3%TFA/ water
B: 0.3%TFA/ methanol
Speed: 1 mL/min
Detection wavelength: 280 nm

Indican median is dribbled onto cotton cloth to moisten, and then dried by adding 500 uL of BGL lysate to allow spontaneous oxidation.

Dye with tyrian purple

Dyeing solutions (DI water 50 mL; tetrahydrofuran 7.5 mL; NaOH 0.5 g; Na2S2O4 0.5 g and chemical 6BrIG 0.15 g or the produced 6BrIG cell dye) are prepared by heating with stirring on the 75-80 °C hot plate for 15 min. After heating, 0.5 g NH4Cl is added to the dyeing solution and subsequent heating is repeated at 75-80 °C. The fabrics (multi-fabric strips or wool fabrics) are soaked into the solution and dye at 60 °C for 15 min.

1.Tryptophan analog responsive variants, 5R and 6R, are transformed into JW4356 E. coli cells.
2.Depending on direct or inverted circuit screening architecture, these are co-transformed with either pNEG+GFP or pPOS+GFP for an inverted signal.
3.Overnight cultures of individual colonies are seeded into a 2 mL 96-well grow block containing 2xYT supplemented with ampicillin, chloramphenicol, and 1 mM IPTG at a 1:20 dilution ratio.
4.Wells contained the indicated concentration of either 5-bromo-L-tryptophan (Sigma) or 6-bromo-L-tryptophan (Gold Biotechnology).
5.Cells are incubated for 5 h at 37 % CO2 to allow expression of repressors and GFP.
6.Prior to fluorescence measurement on a plate reader (TecanM 200, excitation 469 nm; emission 501 nm), cells are centrifuged at 4 °C for 20 min at 3,000 × g and resuspended in 1X PBS solution.
7.Fluorescence intensities are measured and normalized to the OD 600 of the culture. Background JW4356 cell fluorescence is subtracted from all samples, which is calculated by the fluorescence divided by OD 600 for the parental JW4356 strain.
8.Maximal signal output is calculated by transformation of a control vector with wild-type TrpR (for inverter circuit) or inactivated TrpR (for repression circuit).
9.To calculate the half maximal effective concentration (ECs) for repression, the dose-response function for either 5R or 6R is fit to the equation:

where c is the EC50 of 5- or 6-bromo-L-tryptophan (mM),
A is the maximum response,
x is the ligand concentration,
b is the hill coefficient.

Mock selection

1.Both selection plasmids (pPOS and pNEG) are functionally tested by expression of GFP under active or repressed conditions.
2.To test: PLacUV5 drove expression of either a functional TrpR or an inactive TrpR (truncated by two stop codons (K27TAG and Y30TAA) and containing a unique internal HindIII restriction site; plasmid pTrpR.2TAA).
3.To test positive or negative selection CPR, these control constructs are co-transformed with the Taq expressing pPOS or pNEG vectors.
4.A single round of CPR selection is performed (detailed below) for both positive and negative selections using various ratios of active or inactive repressor (1:10 to 1:10,000). Under these conditions, positive selection should enrich for active TrpR repression, whereas negative selection should enrich for inactive TrpR repressors.
5.The enrichment factor for positive selection is estimated to be roughly 300-fold per round of CPR, while the negative selection is estimated to be roughly 200-fold per round of CPR. These are calculated using the following formula:

where (X initial/Y initial) is the initial ratio of active or inactive repressor, EF is the enrichment factor, and (X final/Y final) is the ratio of active or inactive repressor post CPR selection.

CPR directed evolution of biosensors

1.TrpR repressor libraries are created through site-saturation mutagenesis using degenerate oligonucleotides with NNSor NDS codon randomization.
2.Libraries are clonedinto an ampicillin-resistant ColE1 origin plasmid containing the IPTG inducible lacUV5 promoter. Libraries are electroporated into E. coli strain JW4356
(a tryptophan repressor knockout strain) pre-transformed with either the positiveor negative selection vector.
3.Initial library sizes are above the theoretical diversitythreshold (~ 3.0 × 107) and are maintained throughout the CPR selection, witha transformation efficiency of at least 106, but more typically 107-108. Overnightlibrary cultures are diluted 1:20 into fresh 2xYT media supplemented with 100 ug/mL ampicillin, 34 ug/mL chloramphenicol, and 1 mM of 5-bromo-L-tryptophan (Sigma- Aldrich, 99% purity) or 6-bromo-L-tryptophan (Gold
Biotechnology, 99.7% purity), when necessary. Cells are grown for 1 h at 37 °C and subsequently induced by the addition of 1 mM IPTG and incubated at 37 °C for an additional 4 h.
4.Induced cells (200 uL total) are spun in a tabletop centrifuge at 3,000 × g for 8 min. The supernatant is discarded, and the cell pellet is resuspended in 150 uL CPR mix: 1x Taq buffer (10 mM Tris-HCl with pH 8.3, 50 mM KCl, 1.5 mM MgSO4), 260uM dNTPs, 530 nM CPR selection primers (JE.TrpR.CPR.F and JE.TrpR.CPR.R).
5.The resuspended cells are placed into a 2 mL tube with a 1 mL rubbersyringe plunger and 600 uL of oil mix (73% Tegosoft DEC, 7% AbilWE09 (Evonik), and 20% mineral oil (Sigma-Aldrich)).
6.The emulsion is created by placing thecell and oil mix on a TissueLyser LT (Qiagen) with a program of 42 Hz for 4 min.The emulsified cells are thermal-cycled with the program: 95 °C for 3 min, 25 cycles (95 °C for 30 s, 55% CO2 for 30 s, 72 °C for 1 min).
7.Emulsions are broken by spinningthe reaction (10,000 × g for 5 min), removing the top oil phase, adding 150 uL of H2O and 750 uL chloroform, vortexing vigorously, and finally, phase separating in aphase lock tube.
8.DNA in the aqueous phase is cleaned and concentratedusing a PCR purification column. CPR-amplified TrpR genes are isolated through PCR using outnested recovery primers (JE.TrpR.Recov.F and JE.TrpR.Recov.R). Typically, this is achieved by addition of 1/10 the total purified DNA using.
9. Accuprime Pfx (Thermo Fisher) in a 20-cycle PCR; however, challenging rounds ofselection could require increasing the amount of template DNA or cycle number toachieve detectable amplification. Resulting DNA products are re-cloned into theselection vector, completing one full round of CPR selection.