Team:IIT-Madras/Description
Contents
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 any genetic device arises due to various inherent properties of the device. Our study was focused on to address these issues using a device, which was designed to have two genetic components. The first component of the device consisted of the genetic parts (promoters or RBS) required to be characterized were placed in conjunction with a GFP (green florescent protein) producing ORF (open reading frame). Whereas the second component of the device comprised of a RFP (red florescent protein) producing unit placed under fixed promoter. Since the expression level of the second component which consisted of RFP expression would not vary and thus was designed to acts as an internal control. These two genetic components of our device were cloned one after another in the same expression plasmid. The biological parts used in our device for the measurement of inherent noise and how to normalize it to get appropriate results were comprised of different promoters, ribosomal binding sites (RBS), a protein coding parts (ORF) and terminators. Our device was constructed using pSB1A2 plasmid backbone and transformed in E. coli DH5 alpha.
To test the role of RBSs, nature of promoters in giving rise to noise, we made six different devices. All of these six devices had same RFP expressing device (consisting of a constitutive promoter, same RBS, red florescent protein ORF) as internal control. Out of six constructed devices, four GFP expressing devices had same IPTG inducible promoter but variation in RBS parts. Whereas in other two devise, the GFP expressing devices had different constitutive promoters but same RBS.
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Proof
We have successfully constructed and cloned all of these devices. We had estimated cumulative intrinsic and extrinsic noise of all the 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. These experiments helped us to address how we can reduced the impact of these noises in normalization to get accurate result and save us get swayed by erroneous results.
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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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