Motivation

Few decades from now on, we’ll have synthetic bacteria acting like autonomous robots. They’ll produce bioplastics, food, electricity on their own. They’ll be able to precisely detect and differentiate cancer cells from normal cells. Keeping this in mind, we wish to make logic based computations in cells as precise and predictive as we have them in our laptop chips. We seek to apply efficient methods and technology to assess, predict and fix the variability in cells. Considering these things, we have designed our project this year.

If we were to construct a biological oscillator, we would require various genetic parts with different strengths to function like an oscillator. Our oscillator would work only when we make it using parts, which provide complete information about their behavior w.r.t. other parts. Usually, we use a reporter protein to characterize the functionality of device and don’t pay much attention to proper controls, which can lead to erroneous results due to intrinsic and extrinsic factors. This can be called as noise in device.

Noise in Devices

Experimental Design

Noise in device arises due to various inherent properties of the device. Our study is focused to address these issues using a device, which produces two proteins gfp and rfp. Rfp producing unit acts as an internal control and our device of interest, to be characterized, can be placed at gfp producing unit. Most of the biological devices are made up of promoters, RBSs, protein coding parts and terminators. Our device is in pSB1A2 plasmid backbone in E. coli DH5alpha.

To test the role of RBSs, nature of promoters in giving rise to noise, we made six devices. All of these six devices have same RFP expressing device as internal control. Out of six, four GFP expressing devices has same inducible reporter but different RBS parts and in other two, GFP expressing devices has different constitutive promoters.

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Proof

We have successfully cloned all of these devices. We have estimated cumulative intrinsic and extrinsic noise of all six devices. We have observed a trend that intrinsic noise increases as protein expression from device increases The obtained noise data provides us a deeper insight into the rise of noise in devices.

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RIBOS

Design

In order to make precise and predictable biological devices, which can be controlled at will, we have designed a ribo-regulatory switch and named it RIBOS (RNA Inducible Boolean Output like Switch). RIBOS works on the principle of Watson-Crick base pairing between trigger RNA and switch mRNA. RIBOS can be used in place of RBS parts in our expression systems, which can be controlled by supplying trigger RNA molecules. It's applications range from detection and quantification of mRNA molecules to the design of independent and modular genetic circuits in limitless number using forward engineering. As a deliverable, the algorithm can be used by future iGEM teams to design RIBOS-ON and RIBOS-OFF for any trigger sequence.

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Proof

We have sucssefully cloned trigger and switch RNA for RIBOSON and RIBOSOFF devices.

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Notebook

We started working on our project in April. After ideating with our mentors for a few weeks, we drew up a detailed protocol for our project, and the experiments we would need to do to validate. We also maintained a diary where we noted down all our observations and work done everyday. We had quite an eventful summer - the Indian iGEM team meet up happened in July, and we also came up with the idea for our game 'Codonut' and the GM survey during the same time. By August, most of the project work was done, and we began to work on the game and the GM survey.

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