AOX1-α factor-FMO-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1244
Illegal AgeI site found at 2463
- 1000Illegal BsaI site found at 2466
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.
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.
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.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.