Difference between revisions of "Part:BBa K3166000:Experience"
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===Applications of BBa_K3166000=== | ===Applications of BBa_K3166000=== | ||
| + | <p>In order to find a suitable Squalene synthase, we cloned the four Squalene synthase genes from different origins, respectively KSS, NSS, YSS and thSQ & CrtN to the two multiple cloning sites on backbone pETDuet-1 and constructed the plasmid pETDuet-1T7-KSS-2T7-CrtN(pET-KN), pETDuet-1T7-NSS-2T7-CrtN(pET-NN), pETDuet-1T7-YSS-2T7-CrtN(pET-YN) and pETDuet-1T7-thSQS-2T7-CrtN(pET-thN).</p> | ||
| + | <p>To have the Metabolic flux flow towards FPP in the squalene, we overexpressed the genes idi and ispA and added them to plasmid pET-YN, yielding pET-IAY-CN.</p> | ||
| + | <p>Import those five plasmids into E. coli BL21(DE3) to detect the possible formation of carotenoid pigments. By testing the pigmentation level，We discovered that the pigmentation level in the expression of YSS is higher than other strains expressing NSS KSS or thSQS, showing that YSS is relatively active.</p> | ||
| + | https://2019.igem.org/wiki/images/d/d8/T--SEFLS_Shanghai--BBa_K3166000-fig1.png | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 22:17, 21 October 2019
Applications of BBa_K3166000
In order to find a suitable Squalene synthase, we cloned the four Squalene synthase genes from different origins, respectively KSS, NSS, YSS and thSQ & CrtN to the two multiple cloning sites on backbone pETDuet-1 and constructed the plasmid pETDuet-1T7-KSS-2T7-CrtN(pET-KN), pETDuet-1T7-NSS-2T7-CrtN(pET-NN), pETDuet-1T7-YSS-2T7-CrtN(pET-YN) and pETDuet-1T7-thSQS-2T7-CrtN(pET-thN).
To have the Metabolic flux flow towards FPP in the squalene, we overexpressed the genes idi and ispA and added them to plasmid pET-YN, yielding pET-IAY-CN.
Import those five plasmids into E. coli BL21(DE3) to detect the possible formation of carotenoid pigments. By testing the pigmentation level，We discovered that the pigmentation level in the expression of YSS is higher than other strains expressing NSS KSS or thSQS, showing that YSS is relatively active.
User Reviews
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