Project Description

Project Motivation:

Preventing eutrophication

Eutrophication is a worldwide environmental problem which occurs in lakes and reservoirs, often caused by the enrichment of wastewater by the nutrients phosphorus and nitrogen. When this enriched wastewater enters an aquatic environment (which would naturally be low in nutrients), it encourages a vast increase in the growth of algae and other plants, termed an ‘algal bloom’. When these plants inevitably begin to die, decomposition by microorganisms occurs, depleting the dissolved oxygen concentration of the water. The resulting depletion of oxygen means aquatic plants and animals can no longer respire. This can have a devastating effect on biodiversity and ecosystems.

In some cases, eutrophication can also be toxic to human health. Some species of blue green algae (cyanobacteria) release potent toxins, which when consumed even in low concentrations are harmful to human health. This can prevent outdoor activities taking place, for example open-water swimming. Furthermore, predicted climate change and an increasing population may place a further strain on aquatic environments; it is important to minimise the cause of algal blooms where possible.

Wider environmental implications: replacing declining mineral phosphate

Modern intensive agriculture and food security depends on the application of fertilisers: the main components of which are nitrogen, potassium and phosphorus. The limiting component of these fertilisers is phosphate. Mineral phosphorus has to be mined, and therefore is a finite resource; some estimates predict a shortage of mineral phosphate within 100 years. As rock phosphate supplies decline, the cost and demand for phosphate will also rise. It is therefore essential that an alternative to rock phosphate is found. We hope that if successful, the bioremediated phosphate from our bacteria can be recycled and used as fertilisers on fields (see business plan). This will remove the need for mining to take place, which has a damaging effect on the environment. Therefore in future our project may contribute to more sustainable agriculture.

Environmental impact of our product- Phil

Our project builds upon the ideas of enhanced biological phosphate removal (EBPR) in which bacteria known as polyphosphate accumulating organisms (PAOs) in activated sludge acquire phosphate inside their cells. However these current microbiological tools are inefficient and we feel that designing a bacterium to remove phosphate will be much better suited to the task. By creating a bacterium which is a stable accumulator of phosphate, our project aims to prevent the effect of eutrophication at its source. This would provide a more sustainable and economically viable solution to chemical phosphate remediation (see business plan). The byproduct produced by our project, remediated phosphate can be recycled and used as partial replacement to rock phosphate: this may enhance food security and remove the need for damaging phosphate mining.

Who is Phil?

Our team is working on exploiting the natural abilities of Escherichia coli to uptake phosphate. By studying its phosphate metabolism, we aim to improve E. coli‘s phosphate uptake from the environment and therefore engineer a bacterium that can be used as a better alternative to the current methods used by wastewater facilities. For this, our team is looked into the genes responsible for phosphate transport and polyphosphate kinases (PPK) to allow luxury uptake of phosphate into the E. coli cells. We also to borrowed genes from different organisms to enhance the natural bioremediation processes that already exist in our model. Our bacterium should be both efficient and stable for high levels of phosphate uptake.

We envisage our project to be a part of many future applications relating to the uptake and recovery of phosphate, with the possibility of being integrated by industry and with other projects.

Improving a past team's BioBrick:

As part of our construct, we used a BioBrick from Hong Kong University's 2013 iGEM team. BBa_K1217002 is a coding sequence from Kingella oralis known as ppk1 that encodes a polyphosphate kinase. We submitted a new BioBrick BBa_K1807006 that contains HKU's coding sequence that can be used as a KoPPK gene expression device. It has been characterised and sequenced.

In addition we characterised BBa_E0038, a coding sequence for lacZ peptide by including it in our BBa_K1807000 construct. This construct was used to assemble all of our expression devices we made. This part initially had missing functionality on the registry database, and we are now the first iGEM team on record to use this version of the lacZ alpha peptide. We retrieved it from the example database and demonstrated that it is functional for blue-white screening. Initially this biobrick was used in an experiment to make a bifunctional enzymatic and fluorescent reporter of gene expression (Martin et al, 2009). They used the following promoters: BBa_J23119, BBa_J23101, BBa_J23106, BBa_J23115 (in order of decreasing strength) of which all are constitutively active, wheras ours is IPTG inducible. Their ribosome binding site brick was BBa_B0032, which is only 33.96% of BBa_B0034, the BioBrick we used in our construct. In addition, they used a low copy vector- BBa_PSB4A5, the opposite of our high-copy BBa_PSB1C3.

Reference: Martin, Lance, Austin Che, and Drew Endy. "Gemini, a bifunctional enzymatic and fluorescent reporter of gene expression." PLoS One 4.11 (2009): e7569.