Thanks to LINKS_China 2021 team for providing us with part BBa_K4011004(TnaA-FL-FMO) and part BBa_K4011003(Fre-sttH).

We constructed plasmid pET28a-FMO-PtUGT to co-express PtUGT and FMO to convert indole or 6-Br-indole to indican or 6-Br-indican respectively. We also construct plasmid pET28a-BGL and pET28a-FMO to individually express BGL and FMO.

We transform part TnaA-FL-FMO and Fre-sttH into DH5α cell without endogenous TnaA gene. After culture and induced expression of these strains, we use the SDS-PAGE to separate and show the expression of TnaA-FL-FMO and Fre-sttH. We found that we successfully express our fused proteins and these two proteins show high water solubility.

We firstly constructed three plasmids pTrpR, pPOS and pNEG. pTrpR is induced to express muted trp repressors. Active trp repressors with its ligand can induce pPOS to express Taq polymerase and inactive trp repressors or trp repressor without its ligand can induce pNEG to express Taq polymerase.

For GFP test of CPR cycle, we constructed pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase.

For the mock selection of CPR cycle, we inserted a DNA sequence of 200bp into wild type trp repressor to construct INAC-pTrpR (Figure 18). We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. Induced cells were resuspend by CPR mix and emulsion droplets were constructed by mixing CPR mix and oil mix and vortex for 4 min.

We then culture strain that has been induced to express Fre-sttH by NPB solution and 2.5 mM trp and 300 mM NaBr as substrate. During fermentation of 24 h, we use LC-MS to obtained data of concentration of trp and 6-Br-trp by every 6 h. Strain that has been induced to express TnaA-FL-FMO also be cultured by NPB solution and 1 mM trp or 6-Br-trp as substrate. After fermentation for 24 h, we get obvious blue and purple dye in our culture tubes . Besides, we attempt to produce tyrian purple by TnaA-FL-FMO using the culture after fermentation of trp and NaBr with Fre-sttH and the result has no difference from fermentation using standard 1 mM 6-Br-trp.

For purified indigo and tyrian purple, we dissolve the indigo from the cell pellet by DMSO and filter with a 0.2 μm strainer. By diluting DMSO with water, the pigment is precipitated from the solution to obtain a relatively pure indigo solid precipitation. We also use the tyrian purple we produce in cloth dying.

Dye concentration is calculated by a OD610/620 standard curve measured by 2021 iGEM team LINKS_China. We get a ~60% yield for indigo and ~20% yield for tyrian purple.

The expression of Taq polymerase cannot be monitored easily, we apply GFP assay to use GFP production to analyzing the gene circuits function. We also construct pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase. We transform pTrpR which express wild type trp repressor into competent cells that have been transformed with plasmid pNEG-sfGFP or pPOS-sfGFP. Then we culture and induce with different concentration of trp and 1mM IPTG to induce expression of sfGFP. We expect that as trp concentration increases, the expression of sfGFP (RPU/OD600) in pNEG-sfGFP gradually decreases, and the expression of sfGFP (RPU/OD600) in pPOS-sfGFP gradually increases.

In addition to GFP test, we also perform a mock selection to confirm the designed gene circuit function can actually select positive clone. At first, we mix culture with strains that has been induced to express sfGFP and oil mix to check the quality of emulsion droplet. We measure the diameter of droplet with a phase contrast microscopy. We found that the diameter of emulsion droplets are range about 2-10 μm and less than 20% emulsion droplets contain a GFP-expression cell. Thus, we have checked any emulsion droplet can only contain a single cell.

We insert a random sequence into wild type trp repressor to make it inactive to bind to trp, and we get plasmid INAC-pTrpR. We mix plasmid pTrpR with wild type trp repressor and INAC-pTrpR with a mass ratio of 1:1 and transform the mixture into pNEG and pPOS competent cells. A mock CPR seletion is next carried out. Induced strains is emulsified with emulsion mix and replicate positive clone by ePCR. The emulsion remains in a phase-independent state during ePCR. After breaking the emulsion by add ddH2O and chloroform, we apply a recovery PCR and perform a agarose electrophoresis to monitor the result the mock selection. PNEG give an obvious amplification of inactive trp repressor and pPOS give an obvious amplification of active trp repressor.

Our libraries are designed in which residues (I57, V58, R54, L60, G85, T81, I82) that might contact trp at its 6C positions of the indole ring due to their proximity. Considering several position overlapped with those involved in the formation of the HTH motif, we initially choose I57, V58 and T81 as our mutant site for directed evolution. We perform site-directed saturation mutation on these three residues by completely randomized through re-synthesis of the gene with NNS codons (N = A, T, G, C; S = G, C). The library (32768 variants) are transformed into competent cells that have been transformed with pNEG or pPOS beforehand and carry out several circle of CPR to select positive clone we want. Due to the restriction of time, we haven’t perform a selective circle in our project, we plan to simulate this process using mathematical modeling.