Team:BNU-China/Design

Team:BNU-CHINA - 2016.igem.org

DESIGN

Design

This is our design

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).

Fig.0.1 The production of our objective protein

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.

Fig.0.2 The working principle of our protein

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.

1.Expression of α-tubulin、β-tubulin

Taxol plays an important role in mammalian tubulin aggregation, the mainly interaction sites are K19、V23、D26、H227、F270 on β-tubulin[4]. After analyzing the sequence from human breast cancer cell, we determine that tubulin-taxol interactions exist theoretically. Thus we design and synthesize primers based on the sequence of human β-tubulin, adding Hind III and Xho I restrictive sites to the 5' and 3' flanked sites respectively. We extract mRNA from Mcf7 (human breast cancer cell line), obtain cDNAs via reverse transcription and use these as PCR templates to get a-tubulin, β-tubulin respectively.

We ligate α -tubulin, β-tubulin to pET30a(+) vector. The target genes were on the up stream of his-tag and down stream of T7 promoter, and transform the constructed vectors into E.coli TransB(DE3) to express our protein[12][13][14]. After expression, a-tubulin and β-tubulin aggregate spontaneously in vitro[6].

We collaborate with FAFU-CHINA. They help us to verify whether our constructed vectors can express active tubulin monomers in prokaryotic system.

Their team plan to use Co-Immunoprecipitation(CoIP) to confirm their interaction. Since pET30a(+) has His protein tag, they plan to add HA protein tag and Flag protein tag to the down stream of α-tubulin and β-tubulin respectively . The target fragments are amplified by PCR and cloned to T vector and sequenced. Then they link the confirmed gene to pET30a(+) (enzyme site: XhoI, HindIII) .After that，they transform the recombination vector into expression strains(BL21) for culturing. When the concentration of bacterium is appropriate (OD is 0.6-0.8), they induce the strains by 1 mmol IPTG. By using ultrasonic waves to break the cell of bacteria and centrifuging, they obtain the pellet and deal with the inclusion body for the further experiment of Co-Immunoprecipitation(CoIP). （see more in our collaboration.Click to see.）