Difference between revisions of "Part:BBa K4421030"
 
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An inducible caspase-9 suicide gene was placed under the control of a combined UAS-SV40 promoter. The GFP tag serves as a reporter and the 2A peptide sequence was intercalated between iCASP9 and the GFP tag to allow the fusion peptide chain to separate into two peptide chains.  
 
An inducible caspase-9 suicide gene was placed under the control of a combined UAS-SV40 promoter. The GFP tag serves as a reporter and the 2A peptide sequence was intercalated between iCASP9 and the GFP tag to allow the fusion peptide chain to separate into two peptide chains.  
  
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===Special Note===
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To simplify the transfection process and improve the transfection efficiency, we decided to transfect pSV40-UAS-iCASP9-2A-GFP(<partinfo>BBa_K4421030</partinfo>) and NFAT_RE-Gal4-KRAB(<partinfo>BBa_K4421029</partinfo>) on the same plasmid. But the KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB and the forward construct would therefore inhibit the NFAT-RE promoter, we generated the opposite construct, in which the two expression cassettes were cloned in such a manner that both promoters were at opposite ends and at a long distance.
 
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   <li style="display: inline-block;">  [[File:iCASP9-2A-GFP.jpeg|thumb|none|400px|<b></b> pSV40-UAS-iCASP9-2A-GFP]]  
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   <li style="display: inline-block;">  [[File:Circuit section2.jpeg|thumb|none|500px|<b></b> pSV40-UAS-iCASP9-2A-GFP]]  
 
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Latest revision as of 01:44, 7 October 2022


pSV40-UAS-iCASP9-2A-GFP

An inducible caspase-9 suicide gene was placed under the control of a combined UAS-SV40 promoter. The GFP tag serves as a reporter and the 2A peptide sequence was intercalated between iCASP9 and the GFP tag to allow the fusion peptide chain to separate into two peptide chains.

Special Note

To simplify the transfection process and improve the transfection efficiency, we decided to transfect pSV40-UAS-iCASP9-2A-GFP(BBa_K4421030) and NFAT_RE-Gal4-KRAB(BBa_K4421029) on the same plasmid. But the KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB and the forward construct would therefore inhibit the NFAT-RE promoter, we generated the opposite construct, in which the two expression cassettes were cloned in such a manner that both promoters were at opposite ends and at a long distance.

  • pSV40-UAS-iCASP9-2A-GFP

The mechanism and usage of pSV40-UAS-iCASP9-2A-GFP

This part is placed downstream of NFAT_RE-Gal4-KRAB(BBa_K4421029) and the presence of Gal4-KRAB can inhibit the promotor pSV40-UAS(BBa_K4040003), thus diminishing the synthesis of the iCASP9(BBa_K4421002).The engineered cells can survive if the concentration of iCASP9 is low enough after AP1903 addition.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 781
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 463
    Illegal BsaI.rc site found at 1603
    Illegal BsaI.rc site found at 2514