Part:BBa_K3711039
AOX1-α factor-ROX1-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 2460
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1379
Illegal SapI site found at 1638
A kind of yeast protein, which could inhibit promoter Panb1 and then silence following genes of it.
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 band of AOX1-α factor-ROX1-AOX1 Terminator from colony PCR is about 3000bp, identical to the theoretical length of 3070bp estimated by the designed primer location (promoter to terminator), which could demonstrate that this target plasmid had successfully transformed into E.coli.
AOX1 promoter is the strongest eukaryotic promoter currently known in yeast expression system. So we choose AOX1 as the primary promoter when we synthesized all these plasmid for the sake of more convenient expression. But noticing that methanol is hazardous, flammable, combustible and therefore, inappropriate to have direct contact with the hair, we need to substrate AOX1 for constitutive promoter Panb1 and xylose induced promoter Pynr071C to realize the projected regulation function as designed.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
SDS-PAGE
During the test, we find a vague band of ROX1 which couldn’t be verified as successfully expressed according to its SDS-PAGE result in the supernatant. As the key component of our xylose responding system, the successful expression of ROX1 is most vital to our project, so we apply purification through Nickel-affinity chromatography column to ROX1 to further identify its expression. Lucky us, target band is acquired and confirmed. It feels so good to announce that our ROX1 can be expressed like the others.
Different from impure or permeate bands, the target protein located around 55-55kDa, bigger than the theoretical 47.09kDa but still within explainable and acceptable range of glycosylation modification. ROX1 could be confirmed as successfully expressed. Although no gradient elution is applied, following elution result could verify this is ROX1, with some impurity bands occurring in the first ROX1 elution.
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