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 PBLrep (Bba_K2819200)
✔ Characterized expression of flavonoid 3′-hydroxylase (FNS) under arabinose-inducible promoter PBAD (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 PBLrep (BBa_K2819103)
✔ Demonstrated blue light repressible control of luteolin production
STRESS REPORTER
✔ Successfully constructed a stress reporter module
✔ Characterized the burden-responsive promoter PhtpG1 (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 for protein production under PBLrep when OD600 reaches 0.600
To bring ourselves one step closer to actual bioproduction, we scaled up our shake-flask biosynthesis to a 2 L bioreactor. After harvesting the sample, we carried out our safety protocol (see: Safety) and also attempted to extract our target compound, luteolin.
Upon completion of the whole biomanufacturing process, we brought the fruits of our labour back to Ms Leong Minyi, a fashion designer who was a chief source of inspiration for Coup Dy'état, thus coming full circle for our project (see: Integrated Human Practices).
HEAR OUR STORY
We’re using synthetic biology to change the way the world thinks and functions.
We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.
Together we’re going to engineer biology and create something awesome.
CONTACT US
nusigem@gmail.com21 Lower Kent Ridge Rd, Singapore 119077