Difference between revisions of "Team:BNU-China/Design"

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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 <I>E.coli<I/> 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.
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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 <I>E.coli</I> 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.
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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.
 
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.
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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.
 
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.
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</p>
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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 <I>E.coli<I/> Rossatta(DE3) as our expression strain.
+
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 <I>E.coli</I> Rossatta(DE3) as our expression strain.
 
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Thus, we design three groups of parts this year.α-tubulin，β-tubulin expression parts.<ol>
 
Thus, we design three groups of parts this year.α-tubulin，β-tubulin expression parts.<ol>
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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.
 
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.
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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.
 
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.
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Revision as of 17:17, 14 October 2016

Team:BNU-CHINA - 2016.igem.org

MODELING

Modeling

This is our Modeling Design

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.

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.

  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.