W hen amazed that life is a box of chocolate, we basically refer to sweets ignoring the box. However this year we USTC iGEM 2010 Team switch our attention to the unadorned plain box. Our expectation is to construct a Host-cell-free Reaction Chamber using Synthetic Biology methods.
There are always dogmas in Biological field. In text-book, we acquire one that compartmentation is one of the disparate element distinguishing Eukaryotic from Prokaryotic. These compartments guarantee in spatial level the cellular process, from transcription to translation, both individually and collaboratively. Specifically, some membrane-folded organelles, such as Peroxisome, function as barriers to obstacle the diffusion of cellular toxicity,such as hydrogen peroxide. However, recent researches also find out similar accompanying rules in Prokaryotic. Regardless of the membrane-folded organelles, these proteinaceous compartements share several features as their Eukaryotic analogue. Typical for the microcompartments, is one Bacterial Microcompartment (BMC) encoded by pdu operon firstly found in Salmonella enterica. Such BMC restricts 1,2-propanediol metabolism related proteins in the inner lumen that it increases the rate of enzymatic catalytic reactions and sequester propionaldehyde diffuse into the cytoplasm, giving rise to cellular toxic impair.
In this project, we will engineer Escherichia.coil to construct a Host-cell-free Reaction Chamber in three continuous steps. First, we will utilize our bacteria to construct a internal empty BMC by cloning essential pdu shell gene into bacteria. Second, we endeavor with rationale to architect different signal peptides to deliver target enzyme into BMC. Last, we will attempt to improve the traditional purification methods of BMC to enhance the yield of the products.
The advantages of our Host-cell-free Reaction Chamber is obvious. This Chamber could on one hand reduce towards host cells the toxicity yielded from the pathway intermediates, on the other hand subside the influence the host shedding on the synthesis of product, where is the meaning of Host-cell-free coming from. Additionally, Reaction Chamber may enhances the efficiency of the enzyme through the principle of substrates channelling. By achieving all these goals we wish we can construct a robust production line in E.coli.