Difference between revisions of "Team:Jilin China/Improve"

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       <p>This year, we choose GoldenGate Assembly especially for construction. (Learn about our <b>Construction</b>? See our <a href="/Team:Jilin_China/Construction">Construction Page</a>) BsaI and BbsI are used as Type IIs restriction endonuclease in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and better fluorescence intensity. However, sfGFP in iGEM part registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(<a href="http://parts.igem.org/Part:BBa_K2541401">BBa_K2541401</a>) , a BbsI-free sfGFP and especially for GoldenGate Assembly(<b>Fig 1.</b>).  </p>
 
       <p>This year, we choose GoldenGate Assembly especially for construction. (Learn about our <b>Construction</b>? See our <a href="/Team:Jilin_China/Construction">Construction Page</a>) BsaI and BbsI are used as Type IIs restriction endonuclease in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and better fluorescence intensity. However, sfGFP in iGEM part registry (<a href="http://parts.igem.org/Part:BBa_I746916">BBa_I746916</a>) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(<a href="http://parts.igem.org/Part:BBa_K2541401">BBa_K2541401</a>) , a BbsI-free sfGFP and especially for GoldenGate Assembly(<b>Fig 1.</b>).  </p>
 
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       <img src="https://static.igem.org/mediawiki/parts/7/72/SfGFP_BbsI_free-1.svg" width="50%" /></center></html>
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       <img src="https://static.igem.org/mediawiki/parts/7/72/SfGFP_BbsI_free-1.svg" width="80%" /></center>
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       Figure 1. Site mutagenesis for sfGFP BbsI free.
 
       Figure 1. Site mutagenesis for sfGFP BbsI free.
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       <p>Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP (reference，from cxb) without BbsI recognition site. After standardized construction, sfGFP_optimism（BBa_K…,link!）is another choice for our measurement device.
 
       <p>Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP (reference，from cxb) without BbsI recognition site. After standardized construction, sfGFP_optimism（BBa_K…,link!）is another choice for our measurement device.
 
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</p>

Revision as of 08:55, 15 October 2018

IMPROVE

Improve

  • Improvement

    This year, we choose GoldenGate Assembly especially for construction. (Learn about our Construction? See our Construction Page) BsaI and BbsI are used as Type IIs restriction endonuclease in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and better fluorescence intensity. However, sfGFP in iGEM part registry (BBa_I746916) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(BBa_K2541401) , a BbsI-free sfGFP and especially for GoldenGate Assembly(Fig 1.).

    Figure 1. Site mutagenesis for sfGFP BbsI free.

    Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP (reference，from cxb) without BbsI recognition site. After standardized construction, sfGFP_optimism（BBa_K…,link!）is another choice for our measurement device.

    (三种sfGFP的酶切图)

    Firstly, we should know about property of these three types of sfGFP. Here is the result of three-dimensional 荧光检测。。。

    （这里放图）

    For further characterization, we detected the expression of these three types of sfGFP. According to the results, we found out that the expression of sfGFP_optimism is nearly 2.6 as much as the other two types. As a result, we finally choose sfGFP_optimism as our reporter protein.

    More importantly, due to its standardization in GoldenGate Assembly and better property, Part sfGFP_optimism (BBa_K…) is greatly competitive in Best Basic Part.