Team:Tianjin/Design

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Design

This year, we focus on solving the protein purification problem in biological lab. As we talked about in the Protein Extraction Kit page in project description, we aim to replace the lengthy and expensive chromatography with Janus-based aqueous two-phase system. Our design shows many clever points.

Above all, we take advantage of synthetic biology, using the core of iGEM- BioBrick and RFC Assembly Method, to build a universal purification tag. This tag includes a linker containing TEV protease site and sJanus, designed by the RFC23 method which is specific for fusion protein. Thus, if other teams want to use this system to express and purify their proteins, they could use the standard assembly to make a fusion protein.

Secondly, we explore for the best detergent, the best concentration of detergent and the best buffer solution. For example, we tried Berol 532 and Triton-114 as the detergent, and used 2%, 5%, 10%, 20% as the gradient of concentration. We also tried PBS, Tris-HCl and many other buffer solutions. Thirdly, we construct many fusion proteins, including three kinds of fluorescent proteins (GFP, BFP and RFP) with four kinds of Janus, which are inJanus, sJanus and their respective mutants. We also construct experiments about fusion proteins with cutinases Thc_Cut1, FsC and LC to test if this system could be used on the purification of enzymes. After back-extraction, we run SDS-PAGE and calculate the efficiency.

Then we use these data to run our model. To find the universal experiment conditions, we designed a mighty-possible way to do prediction. The first thing to be made clear is that, it is the presence of difference in protein that will affect the effect of extraction most on the first way and then we have to change our experiment conditions to achieve better results. The second thing to know is that, among all properties of protein, the molecular weight, isoelectric point, hydrophobic property is the most possible impact factors, so we build a universal model to find the feasible extraction conditions, such as what concentration we should choose, or how much volume should we add…by choosing the properties of protein as main variables, and the conditions as dependent variables, using the best condition we got from our experiment and model and the protein’s properties as interpolation point to do interpolation, through this way we can gain the formula suits all proteins as long as we know their properties.