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From 2010.igem.org

Parts Design / BioBricks

General summery page here, subpages:

Team:BCCS-Bristol/Wetlab/Part_Design/Components

Team:BCCS-Bristol/Wetlab/Part_Design/Design_Choices

Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks

To make a biosensor one needs a mechanism for sensing and a mechanism for reporting. The sensing mechanism needs to be able to translate a change of concentration in an external chemical to a signal on a genetic level. Many such mechanisms exist naturally, as being able to sense and respond to extra cellular chemicals is an essential part of maintaining homoeostasis.

There are several chemical compounds in fertilizer that could potentially have a corresponding biosensor. Nitrate/nitrite 3 , phosphorous and potassium are the core components of most synthetic fertilizers. Nitrate and potassium play a role in many cellular processes, so it is likely that sensing mechanisms exist for them. The sensor that was designed is nitrate sensitive 4 , this choice was made because there are several potential nitrate sensing pathways in E. coli, as it uses nitrate as an electron acceptor in lieu of oxygen during anaerobic growth.

The sensor works via the principle of a repressed promoter. A promoter is a segment of DNA at the beginning of a gene that attracts the cellular machinery which transcribes genes into mRNA. A promoters strength determines how much transcriptional machinery is recruited, and thus how much mRNA and protein is ultimately produced. A promoter that is ‘repressed’ is being blocked by a repressor, a regulatory protein that reduces its strength. In our case, the repressor is sensitive to nitrates and stops blocking the promoter when it is bound by nitrate molecules (the nitrate is said to be an ‘inducer’). The repressor thus affects gene transcription and may alternately be known as a transcription factor.

When considering the reporting mechanism, the first consideration was the device that is going to record the sensors output. A initial was that the sensor could report visually by eye and a pigment protein could be used as the reporter, but the efficacy of this would be affected by the background colour of the soil and is not necessarily a very quantitative way to report. We briefly considered using a scented protein that could be detected by a trained animal, but again this is not very quantitative. The reporter that was settled on was the family of fluorescent proteins, as fluorescence can be quantitatively measured accurately and cheaply.

These proteins were isolated from marine creatures and fluoresce strongly in response to UV light. Accurate measurements of fluorescence can be made with relatively cheap optical equipment, e.g. a modified digital camera or a spectroscope [7]. The sensor is designed to produce two fluorescent proteins that fluoresce at different wavelengths. A green fluorescent protein (GFP) is produced by a gene that is controlled by the nitrate sensitive promoter, so its concentration is proportional to the amount of nitrate sensed by the cell. A red fluorescent protein (RFP) is produced constitutively 5 , so its concentration is independent of nitrate level. By measuring the level of green fluorescence relative to red fluorescence it is possible to determine the concentration of nitrate without having to normalize for cell density. If only GFP were produced it would be necessary to know the number of cells fluorescing in a given measurement to calculate an accurate value.

It is important to know that nitrate will move from the outside the cell into the cytoplasm where it can interact with the genetic network. It has been shown [8] that during the exponential growth phase E. coli can reach an equilibrium of nitrate to nitrite conversion within 10 minutes. The speed of nitrate uptake decreases in the stationary phase as the expression of the nitrate pump NarK decreases, though it has also been shown that the NarK pump is not essential for nitrate transport into E. coli cells [9]. It is a reasonable assumption that the rate of conversion from nitrate (what is put on the field as fertilizer) to nitrite (a product of nitrate reduction found in the cell) is stable enough to act as a reliable proxy for measurement of nitrate.