Difference between revisions of "Part:BBa K1379001"
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<partinfo>BBa_K1379001 short</partinfo>
 
<partinfo>BBa_K1379001 short</partinfo>
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===Usage and Biology===
  
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SigmaX dependent promoter initiating the transcription of helicase in ''Streptococcus pneumoniae'' NCTC7465 strain. P<sub>comFA</sub> (also known as ''comFAp'') is a promoter which proves to be functional in E. coli and S. pneumonia. The sigmaX protein will bind to 8 base pairs of Phelicase promoter and trigger gene expression. The sigmaX gene and Phelicase promoter used in the construct are both cloned from ''S. pneumoniae'' NCTC 7465 strain. R.P.U (Relative Promoter Unit) of Phelicase is measured to represent promoter strength in reference to constitutive promoter [[Part:BBa_J23101|BBa_J23101]]. Phelicase promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.
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P<sub>comFA</sub> (or <i>comFAp</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 Signal 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>comFA</sub>, alongside with other Com-Box promoters, is turned on by &sigma;<sup>X</sup> and drives expression of the competence protein comFA. (BioCyc)
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== Objective ==
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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>comFA</sub> can only be turned on in the presence of &sigma;<sup>X</sup>.
  
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The objective is to characterize Phelicase promoter so we know whether it iss working in ''E. coli'' DH10B strain or not, and to know the R.P.U (Relative Promoter Unit) with [[Part:BBa_J23101|BBa_J23101]] constitutive promoter as a reference.
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iGEM 2014 Hong_Kong_HKUST Team has cloned P<sub>comFA</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>celA</sub> ([[Part:BBa_K1379000|BBa_K1379000]]), which is another Com-Box promoter recognized by &sigma;<sup>X</sup>, has also been characterized.
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==Method==
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The transcription start site of this promoter has yet to be experimentally located.
  
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By linking Phelicase promoter with GFP generator ([[Part:BBa_E0240|BBa_E0240]]), and SigmaX Generator ([[Part:BBa_K1379006|BBa_K1379006]]), 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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===Characterization===
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==Characterization==
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For characterization, P<sub>comFA</sub> promoter was assembled with the promoter measurement kit [[Part:BBa_E0240|BBa_E0240]] to give the P<sub>comFA</sub> Measurement Kit [[Part:BBa_K1379003|BBa_K1379003]] in plasmid [[Part:pSB3K3|pSB3K3]]. The construct was further assembled with &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]] to give [[Part:BBa_K1379007|BBa_K1379007]].
  
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To measure the RPU (Relative Promoter Unit) of Phelicase 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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Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>comFA</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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The positive control used in this characterization is [[Part:BBa_I20260|BBa_I20260]] which is a constitutive promoter [[Part:BBa_J23101|BBa_J23101]] containing GFP generator [[Part:BBa_E0240|BBa_E0240]], while the negative control used in this characterization is [[Part:BBa_K1379003|BBa_K1379003]] which is Phelicase promoter with GFP generator but without SigmaX generator.
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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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[[File:pcomFA.png|600px|thumb|center|<b>Figure 1. P<sub>comFA</sub> is on in presence but not absence of &sigma;<sup>X</sup>.</b> P<sub>comFA</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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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:pcomFAgraph1.png|500px|thumb|center|<b>Figure 2. P<sub>comFA</sub> has 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</b> P<sub>comFA</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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The above results showed that P<sub>comFA</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:pcomFA.png|600px|thumb|center|'''Figure 1. Phelicase promoter induced by SigmaX protein drives GFP expression. While the same construct without SigmaX protein did not give any GFP signals. Another negative control which is only protein sigmaX without Phelicase also did not give any GFP signals. Reference promoter [[Part:BBa_J23101|BBa_J23101]] + GFP is used as positive control. Scale bar = 5mm.]]
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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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[[File:pcomFAgraph1.png|500px|thumb|center|'''Figure 2. Phelicase promoter Relative Promoter Unit (RPU) is measured with reference to [[Part:BBa_J23101|BBa_J23101]] constitutive promoter. Phelicase promoter induced by SigmaX 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_J23101|BBa_J23101]] promoter strength. Measurement was done by using 3 replicas.]]
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== Discussion ==
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Phelicase promoter is working in ''E. coli'' DH10B strain, and express GFP when induced with SigmaX protein. In comparison to the reference promoter [[Part:BBa_J23101|BBa_J23101]], the R.P.U (Relative Promoter Unit) of PcelA promoter is around 1.2, which is higher than the reference. From Figure 2, we could tell that this promoter is specific as there is almost no GFP expression in the absence of SigmaX protein inducer. This might indicate that the 8 basepairs combox region in Phelicase promoter is specific to SigmaX. However, further characterization needs to be done to evaluate the crosstalk between this inducer-promoter system.
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==Reference ==
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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<br><br><br>
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<partinfo>BBa_K1379001 parameters</partinfo>
 
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===Reference===
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BioCyc was retrieved from http://biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-2079
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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

Revision as of 04:34, 12 October 2014

PcomFA

Usage and Biology

PcomFA (or comFAp) 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 Signal 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. PcomFA, alongside with other Com-Box promoters, is turned on by σX and drives expression of the competence protein comFA. (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 PcomFA can only be turned on in the presence of σX.

iGEM 2014 Hong_Kong_HKUST Team has cloned PcomFA from S. pneumoniae strain NCTC7465 and characterized its Relative Promoter Units (RPU) in presence and absence of a σX generator BBa_K1379006. Promoter PcelA (BBa_K1379000), 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, PcomFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the PcomFA Measurement Kit BBa_K1379003 in plasmid pSB3K3. The construct was further assembled with σX generator BBa_K1379006 to give BBa_K1379007.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the PcomFA 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. PcomFA is on in presence but not absence of σX. PcomFA 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. PcomFA has 1.21 RPU when paired with σX generator. PcomFA 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 PcomFA 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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

BioCyc was retrieved from http://biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-2079

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