Difference between revisions of "Part:BBa K3402051"

 
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<partinfo>BBa_K3402051 short</partinfo>
 
<partinfo>BBa_K3402051 short</partinfo>
  
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This device is composed of two homologous arms upPxa1(BBa_K3402014) and doPxa1(BBa_K3402015), promoter Ptef1(BBa_K3402007), reporter gene yeGFP(BBa_K3402000), NLS(BBa_K3402008), terminator Tsyn7(BBa_K3402001), promoter Pgpd(BBa_K3402006), hygromycin resistance gene hph(BBa_K3402012) and terminator Tcbh2(BBa_K3402020).
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This device is composed of two homologous arms up<i>Pxa1</i>([https://parts.igem.org/Part:BBa_K3402014# BBa_K3402014]) and do<i>Pxa1</i>([https://parts.igem.org/Part:BBa_K3402015# BBa_K3402015]), promoter P<i>tef1</i>([https://parts.igem.org/Part:BBa_K3402007# BBa_K3402007]), reporter gene <i>yeGFP</i>([https://parts.igem.org/Part:BBa_K3402000# BBa_K3402000]), <i>NLS</i>([https://parts.igem.org/Part:BBa_K3402008# BBa_K3402008]), terminator T<i>syn7</i>([https://parts.igem.org/Part:BBa_K3402001# BBa_K3402001]), promoter P<i>gpd</i>([https://parts.igem.org/Part:BBa_K3402006# BBa_K3402006]), hygromycin resistance gene <i>hph</i>([https://parts.igem.org/Part:BBa_K3402012# BBa_K3402012]) and terminator T<i>cbh2</i>([https://parts.igem.org/Part:BBa_K3402020# BBa_K3402020]).
  
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===Usage and Biology===
 
===Usage and Biology===
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We hope our CRISPR/Cas9 system can work in <i>Starmerella bombicola</i> and give play to its high efficiency. Firstly, we constructed a <i>yeGFP</i> expression device, we add nuclear localization sequence (<i>NLS</i>) to the 3’ ending of <i>yeGFP</i> gene and use P<i>tef1</i> as the promoter to express <i>yeGFP</i>. Besides, the hygromycin resistance gene was also built into the plasmid as the marker gene to determine if the transformation is successful.
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<br>
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We set two groups, control group and experimental group, to observe if there is green fluorescence in the nucleus to know if it will work as we suspected. For the control group, the green fluorescence is diffused in the cytoplasm. For the experimental group, we observed the green fluorescence in the nucleus, then this sequence could be used for the subsequent construction of the CRISPR/Cas9 system.
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[[Image:Without NLS.png|500px|thumb|center|Fig.1 Without the <i>NLS</i> (nuclear localization signal peptide)]]
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[[Image:With NLS.png|500px|thumb|center|Fig.2 With the <i>NLS</i> (nuclear localization signal peptide)]]
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Revision as of 02:07, 20 October 2020


yeGFP expression cassette with NLS

This device is composed of two homologous arms upPxa1(BBa_K3402014) and doPxa1(BBa_K3402015), promoter Ptef1(BBa_K3402007), reporter gene yeGFP(BBa_K3402000), NLS(BBa_K3402008), terminator Tsyn7(BBa_K3402001), promoter Pgpd(BBa_K3402006), hygromycin resistance gene hph(BBa_K3402012) and terminator Tcbh2(BBa_K3402020).

Usage and Biology

We hope our CRISPR/Cas9 system can work in Starmerella bombicola and give play to its high efficiency. Firstly, we constructed a yeGFP expression device, we add nuclear localization sequence (NLS) to the 3’ ending of yeGFP gene and use Ptef1 as the promoter to express yeGFP. Besides, the hygromycin resistance gene was also built into the plasmid as the marker gene to determine if the transformation is successful.
We set two groups, control group and experimental group, to observe if there is green fluorescence in the nucleus to know if it will work as we suspected. For the control group, the green fluorescence is diffused in the cytoplasm. For the experimental group, we observed the green fluorescence in the nucleus, then this sequence could be used for the subsequent construction of the CRISPR/Cas9 system.

Fig.1 Without the NLS (nuclear localization signal peptide)
Fig.2 With the NLS (nuclear localization signal peptide)


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2279
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1051
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2279
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2279
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1492