Difference between revisions of "Part:BBa K1379005"
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<partinfo>BBa_K1379005 short</partinfo>
 
<partinfo>BBa_K1379005 short</partinfo>
  
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SigmaX generator consisting constitutive promoter BBa_J23100, RBS BBa_B0034, ''comX'' gene, and double terminator BBa_B0015 which constitutively producing comX protein. Followed by SigmaX inducible PCelA encoding GFP.
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ComX generator consisting constitutive promoter BBa_J23100, RBS BBa_B0034, ''comX'' gene, and double terminator BBa_B0015 which constitutively producing comX protein, followed by ComX inducible PCelA encoding GFP. PcelA (chbBp) is a promoter which proves to be functional in E. coli and S. pneumonia. This promoter works when induced by comX protein. The comX protein will bind to 8 base pairs of PcelA (chbBp) promoter and trigger gene expression. The comX gene and PcelA (chbBp) promoter used in the construct are both cloned from E. coli NCTC 7465 strain. R.P.U (Relative Promoter Unit) of PcelA (chbBp) is measured to represent promoter strength in reference to constitutive promoter BBa_J23100. PcelA (chbBp) promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.
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<br><br>
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'''Objective'''<br>
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The objective is to characterize PcelA promoter so we know whether it’s working in E. coli DH10B strain or not, and to know the R.P.U (Relative Promoter Unit) with BBa_J23100 constitutive promoter as a standard constitutive promoter.
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<br><br>
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'''Method''' <br>
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By linking PcelA promoter with GFP generator (BBa_E0240), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured when the cells are in the mid-log phase. OD595 values were measured and converted to OD600 to obtain the R.P.U of the promoter.
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<br><br>
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'''Characterization'''<br>
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To measure the RPU (Relative Promoter Unit) of PcelA promoter, we adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). With the use of EnVision multilabel reader from Perkin Elmer Company, it enabled us to obtain the fluorescence and absorbance of cells over time. GFP intensity and OD595 values were measured every 30 minutes after the E. coli strains are cultured to mid-log phase.
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<br><br>
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The positive control used in this characterization is BBa_I20260 which is a constitutive promoter BBa_J23100 containing GFP generator BBa_E0240, while the negative control used in this characterization is BBa_K1379002 which is PcelA promoter with GFP generator but without ComX generator.
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<br><br>
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The detailed description including characterization procedure and Data processing of our characterization can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:53, 6 October 2014

σx Generator + PcelA-E0240

ComX generator consisting constitutive promoter BBa_J23100, RBS BBa_B0034, comX gene, and double terminator BBa_B0015 which constitutively producing comX protein, followed by ComX inducible PCelA encoding GFP. PcelA (chbBp) is a promoter which proves to be functional in E. coli and S. pneumonia. This promoter works when induced by comX protein. The comX protein will bind to 8 base pairs of PcelA (chbBp) promoter and trigger gene expression. The comX gene and PcelA (chbBp) promoter used in the construct are both cloned from E. coli NCTC 7465 strain. R.P.U (Relative Promoter Unit) of PcelA (chbBp) is measured to represent promoter strength in reference to constitutive promoter BBa_J23100. PcelA (chbBp) promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.

Objective
The objective is to characterize PcelA promoter so we know whether it’s working in E. coli DH10B strain or not, and to know the R.P.U (Relative Promoter Unit) with BBa_J23100 constitutive promoter as a standard constitutive promoter.

Method
By linking PcelA promoter with GFP generator (BBa_E0240), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured when the cells are in the mid-log phase. OD595 values were measured and converted to OD600 to obtain the R.P.U of the promoter.

Characterization
To measure the RPU (Relative Promoter Unit) of PcelA promoter, we adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). With the use of EnVision multilabel reader from Perkin Elmer Company, it enabled us to obtain the fluorescence and absorbance of cells over time. GFP intensity and OD595 values were measured every 30 minutes after the E. coli strains are cultured to mid-log phase.

The positive control used in this characterization is BBa_I20260 which is a constitutive promoter BBa_J23100 containing GFP generator BBa_E0240, while the negative control used in this characterization is BBa_K1379002 which is PcelA promoter with GFP generator but without ComX generator.

The detailed description including characterization procedure and Data processing of our characterization can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 359
    Illegal BsaI.rc site found at 1490