Difference between revisions of "Part:BBa K3711019"
 
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All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.
 
All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.
  
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   [[File:T--HUST-China--19-2.png|400px|thumb|center|Fig2 Colony PCR result of yeast  
+
   [[File:T--HUST-China--19-2.png|400px|thumb|center|Fig2 Colony PCR result of yeast.]]
 
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.
 
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.
  

Latest revision as of 09:43, 21 October 2021


Panb1


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 124
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Constitutive promoter of yeast. Could initiate expression without inducer. Be inhibited by ROX1.

Molecular cloning

Fig1. Plasmid construction and colony PCR results of reconstructed plasmid with Panb1 and Pynr071C promoter.

All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.

Fig2 Colony PCR result of yeast.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.

SDS-PAGE

Fig3. SDS-PAGE result of FMO after purification of yeast total protein extraction product through Nickel-affinity chromatography column.

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO,.