Design
This is our designOverview
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.
Because the protein sequences we targeted are human breast cell origin, which would 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.Thus, we design three groups of parts this year.α-tubulin，β-tubulin expression parts.
- α-tubulin，β-tubulin expression parts.
- FLC-based fusion protein expression parts.
- 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.
