We applied a two-cell system to produce tyrian purple. Part BBa_K4011004(TnaA-FL-FMO) and part BBa_K4011003(Fre-sttH) from LINKS_China 2021 helped us complete the metabolic processes. We optimized the conditions (time for induction and reaction, concentration of IPTG……) for tyrian production, so that we induced strains to express with high quantity and water solubility and successfully get high yield of tyrian purple (~20%) and indigo (~60%) using Trp and 6-Br-Trp as substrates. We also attempted 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 1mM 6-Br-trp.

We want to co-express FMO and PtUGT by pET28a expression system to convert indole (or 6-Br-indole) to indican. BGL is expressed to acquire solution of enzyme that can convert indican to dye. FMO is also expressed individually to convert indole (or 6-Br-indole) to indigo (or tyrian purple) as a comparison in reaction rate. We construct pET28a-PtUGT-FMO, pET28a-FMO and pET28a-BGL by Gibson assembly and transformed these plasmid into BL21 (DE3). Future project can apply these plasmid for indican production.

The expression of Taq polymerase cannot be monitored easily, we apply GFP assay to use GFP production to analyzing the gene circuits function. We construct pNEG-sfGFP and pPOS-sfGFP which replace Taq polymerase gene with sfGFP to characterize the expression of Taq polymerase. We found that high concentration of trp can inhibit expression of sfGFP in negative selection system.

For mock selection, we inserted a random sequence into wild type trp repressor to make it inactive to bind to trp, and we get plasmid INAC-pTrpR. 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 2~10μm and less than 20% emulsion droplets contain a GFP-expression cell. Thus, we have checked any emulsion droplet can only contain a single cell. And then we mixed 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 cycle was carried out. 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.

We think CPR cycle is a convenient and promising approach in sensor protein directed evoluion.