Team:NUS Singapore/Medal Check List

Awards Check

Measurement Modelling Medal Check List

Team

Team Member Attributions

Collaboration

Collaboration

Human Practice

Silver Up Gold Integrated

Modelling

Overview Methodology Kill Switch for Probiotics Kill Switch for BeeT

Projects

Description Interlab Study Parts Experiment Documentation Improvement Demonstrate Safety Future Works

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Gold Criteria

• Integrated Human Practices

Based on the interview results, we have a better understanding of the potential challenges in building such kill switch and the importance of having kill switches for different synbio applications. From this understanding, we integrate the knowledge gained by proposing building a framework to make engineering customized kill switch easier.

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• Improve a previous part or project

Improved the BBa_K116404 phosphate sensor-GFP reporter constructed by NYMU Taipei in 2008. By replacing the weaker RBS BBA_B0032 with a stronger RBS BBa_B0034, the new promoter is more sensitive to phosphate concentration with a higher expression level.

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• Modelling

We have carried out extensive modelling to design the kill switch for probiotics in silico. We built mathematical models of two kill switch designs (one generated by CELLO and one designed by the team) using ODEs. The performance of kill switch designs was evaluated based on the following criteria: number of parts required in a design, metabolic burden on cell, and response/lag time using ADVANCESYN Studio (a design and modeling software). We ran a number of simulations, what-if studies and sensitivity analysis, and identified the sensitive components in the circuit. Results showed that the kill switch design by the team has faster response and lower metabolic burden. Hence, we proceeded to perform experiments with this design. Apart from the kill switch design for probiotics, we also modelled and studied the blue light–pH NAND gate kill switch for BeeT bacteria from team Wageningen_UR iGEM 2016 using the methodology.

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• Demonstration

We have successfully completed the following tasks:

• Constructed the temperature-sensitive system with GFP reporter (BBa_K2447014）.
• Use modelling to facilitate the design of kill switch.
• Constructed the improved version of the extracellular phosphate sensor with GFP reporter (BBa_K2447000).
• Constructed the blue light activated inducible system with RFP reporter (BBa_K2447501)
• Successfully demonstrated that our proposed standardized methodology for designing kill switches was feasible
• Successfully constructed the plasmid that expressed IM2 protein (anti-toxin) under the control of our phosphate-temperature sensor (BBa_K2447016)
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