Team:Stanford/Research

Our Project

The detection of a ratio of input chemicals is an important biological information processing application that has so far not been realized in a BioBrick standard device or, for the most part, within the larger synthetic biology community. While some sensors have been developed to detect pH (references), such sensors are highly limited in both their application and their output. For our project this year, the Stanford iGEM team decided to design and implement two different varieties of modular ratiometric sensor in order to allow future bioengineers to create more nuanced cellular systems.

The first sensor, which gives a digital readout, uses small RNA interference to calculate the difference in the concentrations of the two input chemicals. One input chemical (chemical A) binds to a promoter and causes the transcription of an mRNA coding for an output protein. The other input chemical (B) binds to a promoter and causes transcription of an sRNA, which is complementary to a target sequence overlapping the ribosome binding site of the mRNA sequence promoted by A. The sRNA then binds to the mRNA, preventing the ribosome from binding and synthesizing the output protein. In the ideal case, no output protein is produced if less A is present than B, and protein begins to be produced as soon as the concentration of A surpasses that of B. To change the threshold ratio detected by the sensor, multiple copies of the genes encoding either the mRNA or the sRNA can be placed downstream of the promoters.

The second sensor uses a transcription factor regulated by a kinase/phosphotase pair in order to provide a more analog readout (detect more than one distinct ratio). In this system, the phosphorylated form of the transcription factor causes transcription of a gene coding for our output protein. The production of the transcription factor is under the control of a constitutive promoter, which maintains a basal concentration. The kinase that acts on the transcription factor is under the control of a promoter positively regulated by A. A phosphotase is similarly controlled by input B. By testing the concentration of output protein in relation to various concentrations of input chemicals, we plan to create an algorithm that will allow us to work backwards from a given concentration of output protein to deduce the ratio of the original concentrations of input chemicals.

Both sensors are modular in that the input and output molecules can be changed without affecting the interior mechanism of the device. We see many applications for this device, including more efficiently regulated metabolic engineering, targeted drug delivery, detection of preterm labor (the ratio of different types of vaginal bacteria has been linked to spontaneous preterm birth, reference), and the discovery of other significant biological ratios.

Project Goals

Digital Sensor

• Accurately report whether the concentrations of input chemicals lie at, above, or below a given ratio
• Allow as little expression as possible at the detection ratio, and as much as possible at all other ratios

Analog Sensor

• Accurately report over a large range of input ratios
• Achieve a linear correlation between output protein concentration and detection ratio
• Have a low basal level of expression

Both Sensors

• Allow future users to change output proteins and input promoters without disrupting the sensor's internal machinery
• Have both sensors be orthogonal to the host system to avoid cross-talk

Important Parameters

Digital Sensor

1. Range of possible ratios
2. Background expression level
   1. Of both proteins at desired ratio
   2. Of either protein when it shouldn't be being expressed
3. Response curve
4. Response time

Analog Sensor

1. Range of possible ratios
2. Background expression level (no kinase-inducing chemical added)
3. Phosphotase specificity
4. Response curve
5. Response time

Research

• Research and Development

• Modeling