Difference between revisions of "Team:NUS Singapore-A/Demonstrate"

We have developed a novel multicomponent biomanufacturing platform, Coup Dy’état, which serves to facilitate the optimization of biomanufacturing. From the various features that distinguish our system, to the heterologous production of compounds, we have successfully demonstrated that each part of our system works as intended. We have also shown how several of the components have been integrated into the biomanufacturing process.

XYLOSE AS FEEDSTOCK

✔ Successfully constructed an inducible xylose-utilizing module

✔ Demonstrated improved growth of E. coli BL21* containing this module in xylose and glucose-xylose mixture

DE NOVO BIOSYNTHESIS

✔ Successfully constructed a naringenin-producing plasmid (with just a single missing enzyme) required for full de novo synthesis

✔ Demonstrated the production of naringenin from coumaric acid in E. coli Acella and BL21*

LUTEOLIN

✔ Successfully constructed chemically-inducible and light inducible luteolin-producing plasmids

✔ Characterized expression of flavonoid 3′-hydroxylase (F3′H) under EL222 blue light repressible promoter P_BLrep (Bba_K2819200)

✔ Characterized expression of flavonoid 3′-hydroxylase (FNS) under arabinose-inducible promoter P_BAD (Bba_K2819206)

✔ Demonstrated the production and extraction of luteolin from naringenin in E. coli BL21*, using shake-flask and bioreactor synthesis (see below)

BLUE LIGHT REPRESSIBLE SYSTEM

✔ Improved characterization of EL222 blue light repressible promoter P_BLrep (BBa_K2819103)

✔ Demonstrated blue light repressible control of luteolin production

STRESS REPORTER

✔ Successfully constructed a stress reporter module

✔ Characterized the burden-responsive promoter P_htpG1 (BBa_K2819010)

✔ Demonstrated that reporter is robust under across different genetic backgrounds and temperatures

✔ Demonstrated that stress was induced by naringenin- and luteolin-producing plasmids, which was detected and reported by the stress reporter module

CELL-MACHINE INTERFACE

✔ Designed and built devices which help characterize optogenetic circuits in petri dishes and 250 ml conical flasks

✔ Designed and built a 500 ml working volume benchtop optogenetic bioreactor, which comprises a peristaltic pump, 2-in-1 OD and fluorescence sensor, and fermentation chamber

✔ Designed and implemented a feedback control system to control the optogenetic bioreactor

✔ Demonstrated the ability of 2-in-1 OD and fluorescence sensor to measure OD₆₀₀ and fluorescence

✔ Demonstrated the ability of the feedback control system to turn off blue light when fluorescence is detected