Team:NUS Singapore/Measurement
Measurement
Overview: Temperature sensitive system
This year, as part of the attempt to implement a kill switch to work in the human GI tract environment, team NUS iGEM 2017 had decided to characterise a new part, which could utilise the ambient temperature as an input. It has always been challenging to characterise temperature-based sensors as the cells’ metabolism would be different in different temperatures. Our work stems from the interesting discovery of the temperature sensitive protein TlpA36 in the virulence plasmid of Salmonella typhimurium by Piraner et al from Caltech. Depending on the temperature, TlpA36 protein can either dimerise (at temperature lower than 36 °C) and block RNA polymerase from binding to the promoter pTlpA, or break into monomers (at temperatures higher than 36 °C) enabling the recruitment of RNA polymerase to bind to pTlpA allowing subsequent downstream gene expression.
As a proof of concept to show that our temperature system BBa_K2447013 was functional, we had constructed an IPTG-inducible temperature sensitive system with a GFP reporter BBa_K2447014, to control the amount of TlpA36 molecules to be produced and to also elucidate the appropriate GFP production (depending on the ambient temperatures). The part is working well in the 10β cells as it had exhibited increasing GFP expression with increasing temperatures (from 30 °C to 37 °C) (Figure 1).
Figure 1: BBa_K2447014 construct which was characterised in 10β cells (incubated overnight). At higher temperatures, the system is switched ‘ON’ as seen by the increase in GFP expression.
Semi-automated characterisation process
iGEM NUS 2017 had sought to utilise automation to speed up the characterisation process. As our team was doing a thermal gradient assay (to characterise GFP expression), all 96 wells in the microplate had to be utilised. If done manually by hand, it would be very tedious to dispense the appropriate inducers and the cells into each well. Furthermore, inconsistency in the dispensing of the appropriate volumes would be introduced due to human error. This is the reason why this year NUS iGEM 2017 employed automation in some of our experiments to speed up the experimental process, reduce the chance of human error being introduced and more importantly, to augment the role of the modern synthetic biologist from a hands-on experimentalist to an overall-system planner. We have utilised Labcyte Echo 525, an automated liquid dispenser to dispense minute volumes of the inducers (from 0 to 1500 nl) and the Opentron liquid handler to dispense higher volumes of cell cultures (from 100 µl to 300 µl) and also to transfer large volumes between multiple 96 well plates. By employing automation, we had significantly improved our experimental workflow by reducing the hands-on experimental time. The Opentron liquid handler is a low cost solution for any SynBio team seeking to speed up their experimental workflow. It is also open source in nature to enable programmers to design their specific dispensing and transferring protocols, and by virtue, it encourages the free exchange of ideas between Synbio groups. It is highly aligned with the iGEM spirit of providing publicly initiated low cost solutions to complex real world problems.
In the near future, we planned to implement a full foundry approach from plasmid construction to characterisation as already seen in major acclaimed universities. In short, our team NUS iGEM 2017 believed that automation is the key for experimentalists of the future to shift their focus from labour intensive on-ground experimentations to that of a meticulous design constructor with high design throughput.
Figure 2: Video of Opentron liquid handler in action transferring liquid from one 96wells plate to another. SynCTI, NUS
Figure 3: Picture of Opentron liquid dispenser (left) and Labcyte Echo 525 liquid dispenser (right). SynCTI, NUS
