Part:BBa_K3711002

ROX1

Rox1 is a repressor that targets the promotor of anb1

A kind of yeast protein, which could inhibit promoter Panb1 and then silence following genes of it. Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1244
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 163
  Illegal SapI site found at 422
  

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.

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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.

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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.

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