Overview

This year our project focuses on in vitro drug screening of anti-cancer medicines which target to tubulin , especially the drugswhich can inhibit the growth of tumor cells by inhibiting the disaggregation of tubulin. The existing methods to extract microtubulin are quite expensive and complex. What’s more, the methods to observe the degree of tubulin aggregation in vitro has some disadvantages such as big error and so on. From this point, we hope to express human tubulin monomers in E.coli prokaryotic expression system and then use FLC (firefly luciferase complementation) or BiFC (bimolecular fluorescence complementation) to detect the aggregation degree of tubulin monomers in vitro easily.So we design a novel system to correctly reclect the aggregation process of microtublin.

Taxol is the most widely used among those anti-cancer drugs. It can inhibit disaggregation and promote aggregation of tubulin. So taxol can inhibit the growth of tumor cells by stabilizing tubulin. Based on this principle, we use our designed novel system to detect the existence of taxol, a kind of widely used anti-disaggregated drug., and hope to quantify taxol concentration by detecting the fluorescence intensity.

In order to achieve this goal, N-luciferase and C-luciferase (or YNE and YCE) are linked to α-tubulin respectively.The three vectors we constructed, n-luc-α-tublin, c-luc-α-tublin, and β-tublin which can express β-tubulin monomer are transformed into E.coli TransB(DE3) competent cells. After purifying α-tubulin linked to N-terminal or C-terminal of reporter protein and β-tubulin, we mix them together in vitro, and then add taxol sample. So we will know taxol or its analogues’ concentration through the fluorescence intensity. And we design a normalized kit as our final product.

Thus, we design three groups of parts this year.α-tubulin，β-tubulin expression parts.

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

As a control of our project, we extract tubulin from porcine brain to explore in vitro tubulin aggregation conditions, and also provide experimental data for modeling.

Moreover, we add the HSP promoter（BBa_K873002）and pBAD promoter（BBa_I0500）to the upstream of phaC1-A-B1 sequence（BBa_K934001）expressing P(3HB) bioplastics, to make the productive process of P(3HB) more controllable.

1. expression of α-tubulin、β-tubulin

Taxol plays an important role in mammalian tubulin aggregation, the mainly binding sites are K19、V23、D26、H227、F270 on β-tubulin. After sequence analysis for human cancer cell, the human breast cancer cell can provide these sites for tubulin-taxol interaction. Thus we design and synthesize primers based on the sequence of human β-tubulin. Meanwhile add Hind III and Xho I restrictive site 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 link a-tubulin, β-tubulin to pET30a(+) vector. The target genes were on the up stream of his-tag and downstream of T7 promoter. After expression, a-tubulin and β-tubulin aggregate spontaneously in vitro.FAFU

Thus we verify that our construct vectors can express active tubulin monomers in prokaryotic system.

2. FLC based fusion protein design

According to previous research, taxol can not only longitudinally stabilize the dimer in protofilament but also laterally stabilize the binding of neighboring protofilaments. Since the taxol interacts with β-tubulin, we design to fuse luciferase fragments to α-tubulin in order to avoid the potential steric effects.

The luciferase was split into two non-overlapping N-terminal (1~416aa) and C-terminal (417~570aa) fragments. When treat with fluorescein B, the mixed expression products could emit fluorescence in 560nm. Thus, we design and synthesize primers based on the sequence of luciferase, and use FLC plasmids pCambia1300-N-Luciferase and pCambia1300-C-Luciferase as PCR templates to get the gene sequence of n-luciferase and c-luciferase.

We design primers to do PCR and adding EcoR I as well as Xho I restriction enzyme sites at the ends of n-luciferase and c-luciferase. We link n-luciferase, c-luciferase to pET30a(+) vector. The target genes are on the up stream of his-tag and down stream of T7 promoter. After we construct the vectors successfully, we transform them into E.coli TransB(DE3) to express thetarget proteins. After purifying proteins using his beads, we mix n-luciferase and c-luciferase together , and then add luciferin B into the system. Microplate reader is used to detect the absorption wavelength at 560 nm. The intensity of background light when they do physical collision in solution can be calculated, and we can verify whether n-luciferase and c-luciferase expressed are active in prokaryotic expression system.

Fusion PCR technology is applied in the further construction of our bio-bricks. The n-luciferase is ligated to N/C terminal of α-tubulin respectively. Similarly, c-luciferase is ligated to N/C terminal of α-tubulin. These four sequences above are the foundation of expressing“α-tubulin-nluc” fusion protein and “α-tubulin-cluc” fusion protein. Moreover, a GGGGSGGGGSGGGS protein linker is also established to the fragment between two target genes. Then, we ligate these fragment to pET30a(+) vector, expressing and purifying our target proteins.

At the same time, we use Gateway large-scale cloning technology to construct nluc-β-tubulin and β-tubulin-cluc gene sequences, ligating them to pET30a(+) expression plasmid for E.coli with restriction endonuclease and T4 ligase methods. They are inserted between T7 promotor and his-tag. Nluc-β-tubulin and β-tubulin-cluc fusion proteins are expressed and purified with the same method mentioned above. Since α-tubulin and β-tubulin can combine as heterodimer spontaneously in vitro, we mix α-tubulin-cluc or cluc-α-tubulin with nluc-β-tubulin, mix α-tubulin-nluc or nluc-α-tubulin with β-tubulin-cluc, and then add luciferin B into the system. Microplate reader is used to detect the absorption wavelength at 560 nm. It can be determined whether n-luciferase and c-luciferase maintain the normal function after fusing with tubulin monomer by observing the fluorescence exists or not.

The proteins who are verified are used for further research. In order to reduce the nonsense collision between protein molecules and avoid background fluorescence, we use semi-solid mixed crowding as the experiment’s buffer. Mixed crowing can modify the inner cellular circumstance. First, we mix α-tubulin-nluc, α-tubulin-cluc and β-tubulin together. They can combine to nluc-tubulin dimer and cluc-tubulin dimer respectively. Since a certain length of microtubule is needed as a premise for taxol’s function, GTP is added into the system to make the heterodimers aggregated into a loci. Because of the aggregation of heterodimer, the nluc and cluc linking to them have a certain possibility to get together and recombine complete luciferase. Luciferin B is added. The intension of fluorescence at the origin length is set as zero point.

We set a series of diluted concentration of taxol as experimental groups. Taxol is mixed with tubulin heterodimer mixture and wait for a certain time, then luciferin B was added. Since the more tubulin heterodimers taking part in the aggregation activity, the more complete luciferase there can be. There would be a relationship between the intension of fluorescence and the length of microtubule. Through modeling, we know that the length of microtubule and the concentration of taxol is positively correlated, so we can fit a standard curve according to the concentration of taxol and the intension of fluorescence detected in the experiment.

3. BiFC based fusion protein design

Except the methods mentioned above, we also use yellow fluorescence protein constructing a similar system based on BiFC theroy.

We split YFP into N-terminal (YNE, amino acids 1~155) and C-terminal (YCE, amino acids 156~240). When the two parts are expressed separately and mixed together in vivo or in vitro, they cannot aggregated into a complete protein, so no fluorescence can be detected. Once the two parts are fusing expressed with a pair of proteins respectively who can interact with each other, YFP can regain its function. The excitation wavelength is 514 nm, emission wavelength is 527 nm. We designed the N-terminal and C-terminal primers based on YFP’s sequence, Using pSPYNE and pSPYCE as templates, running PCR to get YNE and YCE gene sequences.

Then we use the same design as luciferase, both of which serve as the signal proteins. Expression and function verification experiments are carried out later. We used spectrophotometer to detect the intension of emission fluorescence of microtubule under different taxol treatment condition, with excitation wavelength of 527 nm. The standard carve of taxol concentration and fluorescence intension can be measured later.

4. Kit

Based on the design mentioned above, the final product can be carried out as a standardized kit. Our Kit contains these following parts: .

1. The purified α-tubulin-nluc fusion protein, α-tubulin-cluc fusion protein, α-tubulin-YNE fusion protein, α-tubulin-YCE fusion protein and β-tubulin dissolved in mixed crowding respectively.
2. GTP solution with a certain concentration.
3. Luciferase B.
4. User guide.
5. A standard curve for taxol concentration – intensity of excitation fluorescence.

The kit can have various of function: .

1. Quantitative detection to Taxol’s concentration in taxol fermentation broth.
2. Screening microtubule’s anti-depolymerized medicine by detect the intensity of excitation fluorescence after adding the matter be detect to the system.

5. Extract tubulin in virto

Tubulins of animal origin has the ability of self-assembly under proper condition in vitro. When GTP and Mg²⁺ are added into near-neutral pH PIPES buffer, tubulin heterodimers can aggregate into microtubule automatically under room-temperature (or 37 degrees C). This process can be reversed under lower temperature (usual 4 degrees C in experiment condition). It is the basis to extract and purify microtubule in vitro that tubulins can assembly or depolymerize automatically under different temperatures respectively.

As an important member of cytoskeleton, microtubule has extreme important function in organism. Researches focusing on microtubule are of great significance. Porcine brains contain abundant microtubule. As an inexpensive raw material which can be easily purchased, porcine brains are always used for microtubule extraction. Microtubule is conservative in both animal and plant, so the microtubules purified from animal tissues can also be used in plant science research.

To test the aggregation of microtubule effectively, we design the fusion proteins of tubulin subunit and fragments of luciferase and yellow fluorescence protein, respectively. However, whether the fusion proteins can maintain the functions and characters the origin proteins have is largely unknown. So we extract and purifying the porcine origin tubulin, design a series of aggregation and detecting experiment in vitro, in order to provide a positive control for the fusion proteins expressed from prokaryotic cells.