Part:BBa_K1653003
ispA
ispA(from Escherichia coli JM109)
ispA encodes not only geranyl diphosphate synthase but also farnesyl diphosphate synthase.
Farnesyl diphosphate synthase can utilize both dimethylallyl and geranyl diphosphates as substrates, generating geranyl and farnesyl diphosphate, respectively. Therefore the enzyme can catalyze two sequential reactions in the polyisoprenoid biosynthetic pathway.
Usage and Biology
Characterized by SEFLS_SHANGHAI
Construction of the plasmids pET-IAY, pET-IAY-CN, pET-HIAY and pMEP-DG
1. Obtain the target fragments YSS, DXS, ispH, ispG and IIA(rbs-idi-rbs-ispA)
Gene dxs, ispH and ispG were obtained by PCR from E. coli, and IIA fragment was obtained by PCR using plasmid p35151 as template. The following figure shows the fragment YSS, ispH, DXS, ispG and IIA after gel recycling.
2. Conduct double enzyme digestion on the skeleton vectors pBBR1MCS-2, pETDuet-1 and pET-YN, respectively. pBBR1MCS-2 is the skeleton of pMEP-DG, pETDuet-1 is the skeleton of pET-IAY and pET-HIAY, and pET-YN is the skeleton of pET-IAY-CN.
3. The plasmid skeleton recovered by enzyme digestion and the target fragment recovered by gel were verified by electrophoresis. The results are shown below:
4. Connect the homologous recombinant fragment with the corresponding backbone, the ligation reaction system is as follows:
pET-IAY： The enzyme digestion product of pET Duet-1 +IIA+YSS
pET-HIAY： The enzyme digestion product of pET Duet-1 +ispH+IIA+YSS
pET-IAY-CN： The enzyme digestion product of pET-YN + IIA+YSS
pMEP-DG： The enzyme digestion product of pBBR1MCS-2 +dxs+ispG
During transformation, we encountered some difficulties, for example, there were many clones on the plate, but none of them is the positive transformant. Later, we solved this problem by adding DpnI enzyme to the PCR product and speculated that the concentration of plasmid template added was too high when preparing the PCR reaction solution, resulting in excessive negative transformant growing on the plate. We encountered situation where there was no clone on the plate. After increasing the concentration of products from plasmid enzyme digestion and decreasing the concentration of products from gel recycling, this problem is resolved. We speculated that the problem may be due to the low concentration of products from plasmid enzyme digestion and the high concentration of fragment from gel recycling, which affected the accuracy of the ligation reaction system. The addition of plasmids and ligated fragments that are not in the proper range may lead to the failure of ligation reactions.
5. Monoclones on the transformation plate were selected for bacterial solution PCR verification, and the verification results were as follows, indicating that plasmids pET-IAY, pET-IAY-CN, pET-HIAY and pMEP-DG were successfully constructed.
Compare strain H1 and H2
Genes idi and ispA were added to the plasmid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L (strain H2), an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151(Figure. 2). This result indicated that the overexpression of idi and ispA increased the supply of precursor, and thus increasing the yield of squalene.
Figure.2 construction of strains H1, H2 & H3
Compare plasmids pETDuet-1T7-YSS-2T7-CrtN BBa_K3166998 with pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN BBa_K3166808
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, while the pigmentation level in strains containing pET-IAY-CN is 2.2 times that of strains expressing pET-YN, demonstrating that we can significantly increase the metabolic flux towards FPP by expressing idi and ispA.
--> Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 867
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 163
- 1000COMPATIBLE WITH RFC[1000]
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