Difference between revisions of "Part:BBa K3402058"
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===Usage and Biology===
 
===Usage and Biology===
  
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We designed the sgRNA that targeted at <i>Leu</i> site and constructed the <i>PXA1</i>, <i>GFP</i>, <i>Leu</i> triple-gene editing cassette. We transformed it into the recombinant strain. The transformants were cultured on YPD plus hygromycin plates. Then the positive transformants were conducted green fluorescence observation. After that, the positive transformants which lose the function of expressing green fluorescence were dotted on the CM (Complete Medium) and MM (Minimal Medium). The colony which grew on the CM instead of MM were the successfully editing transformants.
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Based on Double-gene editing expression cassette([https://parts.igem.org/Part:BBa_K3402057# BBa_K3402057]), we designed the sgRNA that targeted at Leu site. We constructed the <i>Pxa1</i>, <i>GFP</i>, <i>Leu</i> editing expression cassette, and transformed it into the recombinant strain.
 
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<br>
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The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colonies grown on the CM but did not grow on the MM were the correct transformants.
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[[Image:Triple-gene editing cassette-plate.jpeg|300px|thumb|center|'''Fig. 1''' Green fluorescence observation in fluorescence]]
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[[Image:Colony observation (a CM; b MM).png|500px|thumb|center|'''Fig. 2''' Colony observation '''a)'''Complete Medium '''b)'''Minimal Medium]]
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The triple gene-editing efficiency was 30%.
 
The triple gene-editing efficiency was 30%.
  
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[[Image:Triple-gene editing cassette-plate.jpeg|400px|thumb|center|Most of transformants lose the function of expressing green fluorescence]]
 
  
 
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Revision as of 17:02, 27 October 2020


Triple-gene editing cassette

This device is composed of up-doLeu (BBa_K3402048), sgLeu (BBa_K3402049), 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).

Triple-gene editing cassette.png

Usage and Biology

Based on Double-gene editing expression cassette(BBa_K3402057), we designed the sgRNA that targeted at Leu site. We constructed the Pxa1, GFP, Leu editing expression cassette, and transformed it into the recombinant strain.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colonies grown on the CM but did not grow on the MM were the correct transformants.

Fig. 1 Green fluorescence observation in fluorescence
Fig. 2 Colony observation a)Complete Medium b)Minimal Medium


The triple gene-editing efficiency was 30%.


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 776
    Illegal XhoI site found at 3223
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 174
    Illegal NgoMIV site found at 203
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 3859