Overview

Our project focuses on the drug screening of anti-cancer medicines, especially those that can prevent the growth of tumor cells by inhibiting the disaggregation of microtubule. The existing methods to extract microtubule are quite expensive and complex. What’s more, observing the aggregation or disaggregation level of tubulin requires electron microscope or spectrometer which can measure the light absorption in 350nm. Enormous inconveniences of using such equipment are obvious, not to mean the low accuracy in measurement. Based on current status , we hope to express human tubulin monomers in E.coli prokaryotic expression system, and use FLC (Firefly luciferase complementation) or BiFC (Bimolecular fluorescence complementation) to detect the aggregation degree of tubulin monomers in vitro. Under visible spectrum, the detection should be more easy and sensitive.

Taxol is widely used among anti-cancer medicines. It can inhibit disaggregation therefore stabilize the tubulin^[1][15], preventing the tumor cells from growing. Based on this principle, we plan to use our designed novel system to detect the existence of taxol, and hope to quantify its concentration through fluorescence intensity.

In order to achieve our goal, We ligate N-luciferase and C-luciferase (or YNE and YCE) to α-tubulin respectively for n-luc-α-tublin (YNE-α-tublin) and c-luc-α-tublin (YCE- α-tublin) vectors. We also construct β-tublin vector which can express β-tubulin monomer. All these vectors are transformed into E.coli TransB (DE3) cells for the expression of our objective proteins (Figure 0.1).

After expression and purification of α-tubulin (linked with N/C terminal of signaling proteins) and β-tubulin, we mix them in vitro and add taxol sample. Fluorescence intensity will tell the concentration of taxol or its analogues. (Figure 0.2). Meanwhile, a normalized kit will be designed as our final product.

Because the protein sequences we targeted are from human breast cell, which may have some rare codons. These rare codons may lead to the abnormal expression of tubulin in prokaryote. In order to solve this problem, we use E.coli Rossatta(DE3) as our expression strain ^[2][3].

Our PART-design can be divided into three groups

1. α-tubulin，β-tubulin expression parts.
2. FLC-based fusion protein expression parts.
3. BiFC-based fusion protein expression parts.

As a control group of our project, we extract microtubule from porcine brain to explore the conditions of tubulin aggregation in vitro, which also provide important experimental data to our modeling part.Click to see.