Part:BBa_K3711040

AOX1-α factor-FMO-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 NgoMIV site found at 1244
  Illegal AgeI site found at 2463
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2466
  

Description

This is a composite component for expressing FMO outside the cell. FMO is transcribed and translated into flavin-containing monooxygenase，which is the key enzyme for the synthesis of indigo. It participates in the transformation from indole to indigo. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, FMO is translated and excreted from the cell.

Usage and Biology

Flavin-containing monooxygenase, FMO is one kind of microsomal enzyme widely found in ER of most tissue, whose activity relies on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen. FMO could catalyze the oxidation of most exogenous compounds containing Nitrogen, Sulphur, Phosphorus, Selenium and other nucleophilic elements, which is a vital detoxifying process of carcinogen and many other hazardous substances.
FMO is a kind of monooxygenase, which could produce C4a peroxyflavin intermediate, which is highly stable due to spectrum observation and could remain unchanged under 4℃ for minutes, even hours. In the first cycle, NADPH reduces FAD into FADH2. This reduced flavin could react with oxygen rapidly and become peroxyflavin, which is the main form of FMO and further react wih an appropriate nucleophilic substrate. This reaction will form one molecular of water and transfer one oxygen atom to the substrate. The release of NADP+ may be the rate-limiting step of catalytic cycle while the Vmax of substrate binding has little impact on both reactions.

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.