Difference between revisions of "Part:BBa K3402056"
 
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===Usage and Biology===
 
===Usage and Biology===
  
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We achieve the knockout of <i>PXA1</i> gene and insert the hygromycin resistant gene as a selection marker. Two parts of <i>PXA1</i> are homologous arms at the ends.
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We achieve the knockout of <i>PXA1</i> gene and insert the hygromycin resistant gene as a selection marker. Two parts of <i>PXA1</i> are homologous arms at the ends. The strongest promoter P<i>tef1</i> is used to express sgRNA to guide Cas9 protein to edit <i>PXA1</i> site.
 
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If modified strains can grow on the plate coated with hygromycin, it is successful to knock <i>PXA1</i> with Cas9 system. By comparison, the editing efficiency of the CRISPR/Cas9 system can be clearly observed.
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The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the <i>PXA1</i> site.
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[[Image:Single-gene editing cassette-new.png|500px|thumb|center|'''Fig. 1''' Comparison of the number of transformants using CRISPR/Cas9 and traditional homologous recombination]]
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[[Image:Gel electrophoresis analysis of positive transformants.png|700px|thumb|center|'''Fig. 2''' Gel electrophoresis analysis of positive transformants]]
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'''The single gene-editing efficiency was 100%.'''
  
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[[Image:Single-gene editing cassette.png|500px|thumb|center|The plates contained hygromycin]]
 
  
  

Latest revision as of 16:23, 27 October 2020


Single-gene editing cassette

This device is composed of 50bp-upPXA1(BBa_K3402037), hph(BBa_K3402012), 50bp-doPXA1(BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), Tsyn7(BBa_K3402001).

Single-gene editing cassette sequence.png

Usage and Biology

We achieve the knockout of PXA1 gene and insert the hygromycin resistant gene as a selection marker. Two parts of PXA1 are homologous arms at the ends. The strongest promoter Ptef1 is used to express sgRNA to guide Cas9 protein to edit PXA1 site.
The transformants were cultured on solid YPD medium with hygromycin. Then the genomes of positive transformants extracted for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the PXA1 site.

Fig. 1 Comparison of the number of transformants using CRISPR/Cas9 and traditional homologous recombination
Fig. 2 Gel electrophoresis analysis of positive transformants


The single gene-editing efficiency was 100%.


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 2171
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]