Part:BBa_K3711049
AOX1-α factor-curA-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
This is a composite component for expressing curA outside the cell. CurA is transcribed and translated into NADPH-dependent curcumin / dihydrocurcumin reductase，which is the key enzyme for lycopene degradation. It participates in the transformation from curcumin to curcumin reductase. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, curA is translated and excreted from the cell.
Usage and Biology
curA is derived from curcumin-transforming microorganisms isolated from human faeces with a molecular weight of about 82 kDa and consists of two identical subunits. During the purification of the expressed curA enzyme, the activity of curA enzyme was lost during dialysis. However, we found that the addition of NADPH increased the activity, but the addition of NADH did not increase the activity. These findings indicate that the enzyme catalyzes the NADPH-dependent transformation of curcumin. In the curA catalytic reaction, two steps of curcumin metabolism pathway (curcumin→dihydrocurcumin→ tetrahydrocurcumin) were found. Under the catalysis of CurA, dihydrocurcumin is first the product, then the substrate. These products are produced from curcumin by reducing the diarylheptatrienone chain which could devastate the color of the hair. The curA does not produce more reduction products than tetrahydrocurcumin, which indicates that curA only catalyzes the reduction of compounds with C=C. In the reaction process, the optimum reaction temperature of curA is 35 ℃. The enzyme showed maximum activity at pH 5.9. Although a large number of different compounds have been tested as potential substrates of curA, curA seems to have a narrow substrate spectrum and preferentially acts on curcumin. Considering substrate specificity and NADPH dependence, curA was named NADPH-dependent curcumin/dihydrocurcumin reductase.
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.
The bands of AOX1-α factor-curA-AOX1 Terminator (almost 3000bp), AOX1-α factor-pepACS-AOX1 Terminator (almost 2000bp) and AOX1-α factor-DsbC-AOX1 Terminator (almost 3000bp) from colony PCR are identical to the theoretical lengths of 2875bp, 1987bp and 2722bp 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.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
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