Part:BBa_K3279000

Geranylgeranyl diphosphate synthase (CrtE) from Rhodobacter sphaeroides

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 852
  
• 1000
  INCOMPATIBLE 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.

Fig.1 The site-specific mutation of CrtE.