Difference between revisions of "Part:BBa K1379005"
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__NOTOC__
 
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<partinfo>BBa_K1379005 short</partinfo>
 
<partinfo>BBa_K1379005 short</partinfo>
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<br> This is P<sub>celA</sub> Measurement Kit [[Part:BBa_K1379002|BBa_K1379002]] assembled with &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]].
  
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ComX generator consisting constitutive promoter [[Part:BBa_J23100|BBa_J23100]], RBS ([[Part:BBa_B0034|BBa_B0034]]), ''comX'' gene, and double terminator [[Part:BBa_B0015|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 [[Part:BBa_J23100|BBa_J23100]]. PcelA (chbBp) promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.
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===Usage and Biology===
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<br><br>
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'''<font size="4">Objective</font>'''<br>
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P<sub>celA</sub> (or <i>celAp</i>) is a &sigma;<sup>X</sup> regulated promoter from <i>Streptococcus pneumoniae</i>.  It is a member of the Cin-Box or Com-Box promoter family that share the 8 base pair consensus sequence of TACGAATA, where the sigma factor &sigma;<sup>X</sup> [[Part:BBa_K1379004|BBa_K1379004]] (or ComX) binds to and promotes transcription initiation. (Piotrowski, Luo, & Morrison, 2009) During the exponential growth of <i>S. pneumoniae</i>, Competence Stimulating Peptide (CSP) mediated quorum sensing induces expression of &sigma;<sup>X</sup>. &sigma;<sup>X</sup> as a global regulator then directs <i>S. pneumoniae</i> to enter a transient competent cell state. P<sub>celA</sub>, alongside with other Com-Box promoters, is turned on by &sigma;<sup>X</sup> and drives expression of the competence protein CelA. (BioCyc)
  
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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 [[Part:BBa_J23100|BBa_J23100]] constitutive promoter as a standard constitutive promoter.
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Voigt and his colleagues have demonstrated that orthogonal gene expression could be achieved through the use of &sigma;s, anti-&sigma;s and synthetic promoters. (Rhodius et al., 2013). This principle has been demonstrated to work in <i>E. coli</i> (see characterization below), in which P<sub>celA</sub> can only be turned on in the presence of &sigma;<sup>X</sup>.
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<br><br>
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'''<font size="4">Method</font>''' <br>
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iGEM 2014 Hong_Kong_HKUST Team has cloned P<sub>celA</sub> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its Relative Promoter Units (RPU) in presence and absence of a &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]]. Promoter P<sub>comFA</sub> ([[Part:BBa_K1379001|BBa_K1379001]]), which is another Com-Box promoter recognized by &sigma;<sup>X</sup>, has also been characterized.
  
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By linking PcelA promoter with GFP generator ([[Part:BBa_E0240|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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The transcription start site of this promoter has yet to be experimentally located.
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<br><br>
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'''<font size="4">Characterization</font>'''<br>
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===Characterization===
  
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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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For characterization, [[Part:BBa_K1379005|BBa_K1379005]] which contain P<sub>celA</sub> Measurement Kit [[Part:BBa_K1379002|BBa_K1379002]] assembled with &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]] in plasmid [[Part:pSB3K3|pSB3K3]] was used.
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<br><br>
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The positive control used in this characterization is [[Part:BBa_I20260|BBa_I20260]] which is a constitutive promoter [[Part:BBa_J23100|BBa_J23100]] containing GFP generator [[Part:BBa_E0240|BBa_E0240]], while the negative control used in this characterization is [[Part:BBa_K1379002|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].
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Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter [[Part:BBa_J23101|BBa_J23101]],  [[Part:BBa_I20260|BBa_I20260]] was used as a positive control; [[Part:BBa_E0240|BBa_E0240]] was used as the negative control for background fluorescence.
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[[File:pcelAphoto1.png|600px|thumb|center|<b>Figure 1. P<sub>celA</sub> is on in presence but not absence of &sigma;<sup>X</sup>.</b> P<sub>celA</sub> Measurement Kit gave strong GFP signals comparable to [[Part:BBa_I20260|BBa_I20260]] when assembled without &sigma;<sup>X</sup> generator, and fluorescence was not observed with the same construct without the &sigma;<sup>X</sup> generator. Scale bar = 5mm.]]
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<br>
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Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an <i>in vivo</i> reference standard” (Kelly et al., 2009).  <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter [[Part:BBa_J23101|BBa_J23101]] measurement device [[Part:BBa_I20260|BBa_I20260]] to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.
 
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[[File:pcelAgraph1.png|500px|thumb|center|<b>Figure 2. P<sub>celA</sub> has 0.53 RPU when paired with &sigma;<sup>X</sup> generator.</b> P<sub>celA</sub> was measured in reference to [[Part:BBa_J23101|BBa_J23101]] constitutive promoter with and without &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]]. RPU shown was calculated from 3 replicas.]]
  
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[[File:only_pcelAA.png|500px|thumb|center|'''Figure 1. PcelA and Phelicase promoter induced by com-X protein drives GFP expression. PcelA and Phelicase promoter induced by com-X protein gave GFP signals. While the same construct without com-X protein did not give any GFP signals. Another negative control which is only protein comX without PcelA/Phelicase also did not give any GFP signals. Reference promoter [[Part:BBa_J23100|BBa_J23100]] + GFP is used as positive control. Scale bar = 5mm.]]
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The above results showed that P<sub>celA</sub> is functional in <i>E. coli</i> and is turned on specifically in presence of &sigma;<sup>X</sup>. Further characterization can be done to evaluate crosstalk between this sigma factor-promoter pair and their other counterparts.
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[[File:pcelAgraph.png|500px|thumb|center|'''Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to [[Part:BBa_J23100|BBa_J23100]] constitutive promoter. PcelA promoter induced by com-X protein gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on [[Part:BBa_J23100|BBa_J23100]] promoter strength. Measurement was done by using 3 replicas.]]
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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Extensive documentation of this characterization, including the wet lab protocols and data processing, can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1379005 parameters</partinfo>
 
<partinfo>BBa_K1379005 parameters</partinfo>
 
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===Reference===
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BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867
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<br><br>
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Luo P., & Morrison D. (2003).'' Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae''. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003
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<br><br>
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Piotrowski A., Luo P., & Morrison D. (2009). ''Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.'' Journal of Bacteriology. doi:10.1128/JB.01750-08
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<br><br>
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Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). ''Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.'' Molecular Systhetic Biology .doi:10.1038/msb.2013.58
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<br><br>
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J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3, 4. doi: 10.1186/1754-1611-3-4

Latest revision as of 13:47, 17 October 2014

σx Generator + PcelA-E0240
This is PcelA Measurement Kit BBa_K1379002 assembled with σX generator BBa_K1379006.

Usage and Biology

PcelA (or celAp) is a σX regulated promoter from Streptococcus pneumoniae. It is a member of the Cin-Box or Com-Box promoter family that share the 8 base pair consensus sequence of TACGAATA, where the sigma factor σX BBa_K1379004 (or ComX) binds to and promotes transcription initiation. (Piotrowski, Luo, & Morrison, 2009) During the exponential growth of S. pneumoniae, Competence Stimulating Peptide (CSP) mediated quorum sensing induces expression of σX. σX as a global regulator then directs S. pneumoniae to enter a transient competent cell state. PcelA, alongside with other Com-Box promoters, is turned on by σX and drives expression of the competence protein CelA. (BioCyc)

Voigt and his colleagues have demonstrated that orthogonal gene expression could be achieved through the use of σs, anti-σs and synthetic promoters. (Rhodius et al., 2013). This principle has been demonstrated to work in E. coli (see characterization below), in which PcelA can only be turned on in the presence of σX.

iGEM 2014 Hong_Kong_HKUST Team has cloned PcelA from S. pneumoniae strain NCTC7465 and characterized its Relative Promoter Units (RPU) in presence and absence of a σX generator BBa_K1379006. Promoter PcomFA (BBa_K1379001), which is another Com-Box promoter recognized by σX, has also been characterized.

The transcription start site of this promoter has yet to be experimentally located.

Characterization

For characterization, BBa_K1379005 which contain PcelA Measurement Kit BBa_K1379002 assembled with σX generator BBa_K1379006 in plasmid pSB3K3 was used.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the PcelA Measurement Kits with and without the σX generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, BBa_I20260 was used as a positive control; BBa_E0240 was used as the negative control for background fluorescence.

Figure 1. PcelA is on in presence but not absence of σX. PcelA Measurement Kit gave strong GFP signals comparable to BBa_I20260 when assembled without σX generator, and fluorescence was not observed with the same construct without the σX generator. Scale bar = 5mm.


Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). E. coli DH10B strains holding the constructs with or without σX generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter BBa_J23101 measurement device BBa_I20260 to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.

Figure 2. PcelA has 0.53 RPU when paired with σX generator. PcelA was measured in reference to BBa_J23101 constitutive promoter with and without σX generator BBa_K1379006. RPU shown was calculated from 3 replicas.

The above results showed that PcelA is functional in E. coli and is turned on specifically in presence of σX. Further characterization can be done to evaluate crosstalk between this sigma factor-promoter pair and their other counterparts.

Extensive documentation of this characterization, including the wet lab protocols and data processing, 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


Reference

BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867

Luo P., & Morrison D. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003

Piotrowski A., Luo P., & Morrison D. (2009). Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW. Journal of Bacteriology. doi:10.1128/JB.01750-08

Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Molecular Systhetic Biology .doi:10.1038/msb.2013.58

J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3, 4. doi: 10.1186/1754-1611-3-4