Plasmid; Resistant medium with antibiotics; HEPES solution; NaCl solution; Imidazole solution; Protein buffer; Chloroform; Ventilation cabinet; Ethyl acetate; Tryptophan; 6-chloro-tryptophan; 6-bromotryptophan, Methanol.

Reagents

Equipments

Centrifuge tube; 37 ℃ shaker; OD measurement instrument; centrifuge; Ultrasonic instrument; Filter screen.

Procedure

(1)Expression, purification and reaction of UGT-FMO

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.

(2)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.

Equipments

Emulsion PCR

Spectrophotometer 1 mL Syringes; TissueLyser; Microman pipette for viscous liquids; Capillary pistons; Inverted Epi-Fluorescence Phase Contrast Microscope; FITC/GFP filter cube; Cellometer cell counting chambers; Microscope camera calibration slide; ImageJ.

Procedure

Centrifuge tube; 37 ℃ shaker; OD measurement instrument; centrifuge; Ultrasonic instrument; Filter screen.

Procedure

(1)GFP Test
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.

(2)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.

Reagents

Equipments

Spectrophotometer 1 mL Syringes; TissueLyser; Microman pipette for viscous liquids; Capillary pistons.

Procedure

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.