Difference between revisions of "Team:Jilin China/Improve"
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| − | + | <h2>Improvement</h2> | |
| + | <p>This year, we choose GoldenGate Assembly especially for construction. (Learn about our Construction? Link!) 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,link!) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to finish a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP_for GoldenGate Assembly part(BBa_K…,link!) , a BbsI-free sfGFP and especially for GoldenGate Assembly. </p> | ||
| + | <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> | ||
</div> | </div> | ||
</li> | </li> | ||
Revision as of 08:32, 8 October 2018
IMPROVEImprove
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Improvement
This year, we choose GoldenGate Assembly especially for construction. (Learn about our Construction? Link!) 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,link!) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to finish a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP_for GoldenGate Assembly part(BBa_K…,link!) , a BbsI-free sfGFP and especially for GoldenGate Assembly.
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.