Difference between revisions of "Part:BBa K3402057"
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===Usage and Biology===
 
===Usage and Biology===
  
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We knock out the <i>PXA1</i> and <i>GFP</i> gene by this device. In the meantime, we insert <i>hph</i> gene at the origin <i>PXA1</i> site by homology directed repair, which is to use <i>hph</i> as the marker gene to know if it is successful to knock <i>PXA1</i>.  
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We knocked out the <i>PXA1</i> and <i>GFP</i> gene by this device. In the meantime, we inserted hygromycin resistance gene at the origin <i>PXA1</i> site by homology directed repair, which was to used as the marker gene to know if it is successful to knock <i>PXA1</i>.  
 
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At last, we observe the strains that growed on the plate coated with hygromycin and check if strains can also lose the function of expressing green fluorescence. Then, we get the conclusion that the success of double gene editing was proved.
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The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were extracted genomes for PCR, gel electrophoresis analysis and conducted green fluorescence observation. As a result, all of the target fragments were verified to be right in gel electrophoresis analysis and only two positive colonies showed fluorescence.
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The double gene-editing efficiency was 99%.
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[[Image:Double-gene editing cassette-plate.jpeg|400px]]
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[[Image:Double-gene editing cassette-GFP.png|500px|thumb|center|'''Fig. 1''' Green fluorescence observation in fluorescence]]
 
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'''Fig. a)'''Strains can grow on the plate coated with hygromycin. '''b)'''Most of strains also lose the function of expressing green fluorescence.
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[[Image:Double-gene editing-gel.png|500px|thumb|center|'''Fig. 2''' Gel electrophoresis analysis of positive transformants]]
  
  

Revision as of 16:46, 27 October 2020


Double-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), sgGFP(BBa_K3402024), Tsyn7(BBa_K3402001), upGFP(BBa_K3402025), doGFP(BBa_K3402026).

Double-gene editing cassette.png

Usage and Biology

We knocked out the PXA1 and GFP gene by this device. In the meantime, we inserted hygromycin resistance gene at the origin PXA1 site by homology directed repair, which was to used as the marker gene to know if it is successful to knock PXA1.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were extracted genomes for PCR, gel electrophoresis analysis and conducted green fluorescence observation. As a result, all of the target fragments were verified to be right in gel electrophoresis analysis and only two positive colonies showed fluorescence.


Fig. 1 Green fluorescence observation in fluorescence


Fig. 2 Gel electrophoresis analysis of positive transformants


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2807