Difference between revisions of "Part:BBa K3279000"
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There is an illegal site (PstI) in the original sequence of CrtE (see Figure 1a). In order to remove the illegal site, we designed two primers matching with the region but change the PstI site (CTGCAG) into CTGCAA (see Figure 1b). Then we amplified CrtE into two parts (see Figure 1c, 1d) and then linked them again using overlap PCR (see Figure 1e). In this way we removed the illegal site but did not change the amino sequence. But we finished this job too late, leading us no time to cloned our engineered CrtE into the plasmid pACYC184-M, which was a pity. | There is an illegal site (PstI) in the original sequence of CrtE (see Figure 1a). In order to remove the illegal site, we designed two primers matching with the region but change the PstI site (CTGCAG) into CTGCAA (see Figure 1b). Then we amplified CrtE into two parts (see Figure 1c, 1d) and then linked them again using overlap PCR (see Figure 1e). In this way we removed the illegal site but did not change the amino sequence. But we finished this job too late, leading us no time to cloned our engineered CrtE into the plasmid pACYC184-M, which was a pity. | ||
| − | [[File:CAU CrtE Fig1.png|800px|thumb|center| | + | [[File:CAU CrtE Fig1.png|800px|thumb|center|Fig.1 The site-specific mutation of CrtE.]] |
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Latest revision as of 11:34, 20 October 2019
Geranylgeranyl diphosphate synthase (CrtE) from Rhodobacter sphaeroides
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 852
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 531
Illegal SapI.rc site found at 592
This part is a geranylgeranyl diphosphate synthase which transforms (2E,6E)-farnesyl diphosphate into geranylgeranyl diphosphate. It is a crucial enzyme in lycopene synthesis pathway and always works together with phytoene synthase (CrtB) and phytoene desaturase (CrtI). This sequence is optimized for use in E.coli.
Usage and Biology
There is an illegal site (PstI) in the original sequence of CrtE (see Figure 1a). In order to remove the illegal site, we designed two primers matching with the region but change the PstI site (CTGCAG) into CTGCAA (see Figure 1b). Then we amplified CrtE into two parts (see Figure 1c, 1d) and then linked them again using overlap PCR (see Figure 1e). In this way we removed the illegal site but did not change the amino sequence. But we finished this job too late, leading us no time to cloned our engineered CrtE into the plasmid pACYC184-M, which was a pity.
