Abstract

Overloading of phosphorus and nitrogen are the most important causes of red tide. So it is necessary to construct corresponding sensors able to respond effectively and accurately. Once our design is finished, it could be integrated into our information-processing devices, a comparator or ratio sensor. We have investigated a lot about the endogenous phosphate sensor and nitrate sensor in prokaryotes. Even though we successfully screened some candidates for further modification, it is still frustrating that few mechanisms match perfectly with the corresponding phosphate and nitrate concentration when red tide probably occurs. So fine-tuning of nitrate and phosphate sensor is somewhat difficult but necessary. Given the limited time, we have focused more on the promoter test platform construction and characterization. Since a variety of sensors rely on the two-component system(TCS) to function, we also conceive a whole set of design ideas for TCS modifications, thereby enable other teams to fine-tune their sensors to respond desirably. In addition，in order to not only construct corresponding sensors to predict the red tide, but also prevent it from exploding, we did job in searching biological phosphate accumulation.

Background

Overview of two-component system

To adapt and survive in different microenvironments, bacteria must sense and respond to extracellular signals. The adaptive response to environmental stimuli can be transduced by two-component regulatory systems, which are involved in the regulation of chemotaxis, osmoregulation, metabolism and transport. A typical TCS is comprised of a membrane-bound histidine kinase (HK) and a partner response regulator (RR). Specific environmental signals lead to activation (autophosphorylation) of the HK, which in turn acts as a phosphoryl donor for activation (autophosphorylation) of the partner RR. Phosphate and nitrate sensors[1].

Phosphate sensor----phoBR system[1]

The extracellular concentration of phosphate is sensed by the two-component regulatory system PhoBR, in which PhoR encodes the HK and PhoB the RR. PhoB, when phosphorylated by PhoR, would form homodimers and bind to the consensus pho-box sequence involved in the corresponding promoter and activate downstream genes expression. Generally speaking, phoBR system responds to phosphate limitation, when the extracellular phosphate concentration falls below 4 uM. Though iGEM-2008 team of NYMU-Taipei has submitted the information of that part BBa_K116401, we couldn’t get it from MIT registry. So we have decided to acquire and characterize this part by ourselves.

Nitrate-sensitive sensor----NarXL system[2]

Similar to phoBR system, NarX serves as the trans-membrane HK and NarL serves as the RR. Once the extracellular concentration of nitrate reaches certain concentration, NarX autophosphorylates and acts as the phosphate group donor of NarL. Two phospharylated NarL molecules form a homodimer and act as DNA-binding proteins that activate the transcription of downstream genes expression. The nitrate-sensitive promoter is well-characterized by Edinburgh University iGEM Team 2009 and we plan to fine-tune that part in order to function desirably when applied to our design.

phosphate accumulation[3]

An enzyme responsible for polyP synthesis is the homotetrameric polyP kinase (PPK) ppk polymerizes the terminal phosphate of ATP into polyp. Acetate kinase (ackA) was employed as an ATP regeneration system for polyphosphate synthesis. Researchers have demonstrated Strain MV1184 containing ppk and ackA showed the highest rates of Pi removal from the medium.