Part:BBa_K3711042

AOX1-α factor-crtE-AOX1 Terminator

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1187
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1863
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

This is a composite component for expressing crtE outside the cell. CrtE is transcribed and translated into Geranylgeranyl pyrophosphate synthase，which is the key enzyme for the synthesis of curcumin. It participates in the transformation from Farnessee pyrophosphate (FPP) to Geranylgeranyl pyrophosphate (GGPP). AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, crtE is translated and excreted from the cell.

Usage and Biology

CrtE is derived from Erwinia, encodes Geranylgeranyl pyrophosphate synthase and participates in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two molecules of GGPP into octahydrolycopene and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. The crtE encodes Geranylgeranyl pyrophosphate synthase which synthesizes GGPP. Laboratory studies have shown that E.coli transformed with E.herbicola carotenoid synthesis gene could resist higher level of LTV radiation and phototoxic environment, indicating that the synthesis of carotenoid may be very important for the survival of E.herbicola in nature.

Molecular cloning

Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.

Figure1:Colony PCR results of AOX1-α factor-FMO-AOX1 Terminator, AOX1-α factor-crtE-AOX1 Terminator, AOX1-α factor-crtB-AOX1 Terminator and AOX1-α factor-crtI-AOX1 Terminator transformed E.coli.

The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1 Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Figure2:Colony PCR result of yeast after electroporation through electrophoresis.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.