Difference between revisions of "Part:BBa K1379004"
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===Usage and Biology===
 
===Usage and Biology===
  
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&sigma;<sup>X</sup> (ComX) is an alternative &sigma; factor in <i>Streptococcus pneumoniae</i> which serve as a competence-specific global transcription modulator. (BioCyc) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the Competence Signal Peptide (CSP). (Luo & Morrison, 2003) Upon stimulation by CSP, &sigma;<sup>X</sup> will be expressed and associate with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>X</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. (Piotrowski, Luo, & Morrison, 2009)
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&sigma;<sup>X</sup> (ComX) is an alternative &sigma; factor in <i>Streptococcus pneumoniae</i> which serves as a competence-specific global transcription modulator. (BioCyc) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the Competence Stimulating Peptide (CSP). (Luo & Morrison, 2003) Upon stimulation by CSP, &sigma;<sup>X</sup> will be expressed and associate with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>X</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>X</sup> were found to contain a 8-bp consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009)
  
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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). The principle depicted applies to &sigma;<sup>X</sup>, which when paired with the promoters it regulates, achieves orthogonal expression in <i>E. coli</i>. It should be noted that iGEM 2014 Hong_Kong_HKUST Team has compared the amino acid sequences of ECFs described by Rhodius et al. with that of &sigma;<sup>X</sup> from <i>S. pneumoniae</i>, and found no significant similarities, thus &sigma;<sup>X</sup> is a new sigma factor added to the list.
  
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iGEM 2014 Hong_Kong_HKUST Team has cloned &sigma;<sup>X</sup> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters: P<sub>chbB</sub>([[Part:BBa_K1379000|BBa_K1379000]]) and P<sub>comFA</sub> ([[Part:BBa_K1379001|BBa_K1379001]]).
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iGEM 2014 Hong_Kong_HKUST Team has cloned &sigma;<sup>X</sup> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters: P<sub>celA</sub>([[Part:BBa_K1379000|BBa_K1379000]]) and P<sub>comFA</sub> ([[Part:BBa_K1379001|BBa_K1379001]]).
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Following the principle depicted in Rhodius et al., (Rhodius et al., 2013) &sigma;<sup>X</sup> could achieve orthogonal gene expression regulation if used together with other sigma factors / ECFs. It should be noted that iGEM 2014 Hong_Kong_HKUST Team has compared the amino acid sequences of ECFs described by Rhodius et al. with that of &sigma;<sup>X</sup> from <i>S. pneumoniae</i>, and found no significant similarities, thus &sigma;<sup>X</sup> is a new sigma factor added to the list.
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===Characterization===
 
===Characterization===
  
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To test the functionality of &sigma;<sup>X</sup>, we first enabled constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator [[Part:BBa_K1379006|BBa_K1379006]]. The generator was then assembled with the promoter measurement kit [[Part:BBa_E0240|BBa_E0240]] with either promoter P<sub>chbB</sub> (Promoter only: [[Part:BBa_K1379000|BBa_K1379000]], w/ BBa_E0240: [[Part:BBa_K1379002|BBa_K1379002]]) and P<sub>comFA</sub> (Promoter only: [[Part:BBa_K1379001|BBa_K1379001]], w/ BBa_E0240: [[Part:BBa_K1379003|BBa_K1379003]]). <i>E. coli</i> colonies holding the resulting constructs in [[Part:pSB3K3|pSB3K3]] were observed under 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 general negative control. for background fluorescence. Measurement kits for P<sub>chbB</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.
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To test the functionality of &sigma;<sup>X</sup>, we first enabled constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator [[Part:BBa_K1379006|BBa_K1379006]]. This generator, which expresses &sigma;<sup>X</sup>, was assembled with the promoter measurement kit [[Part:BBa_E0240|BBa_E0240]] with either promoter P<sub>celA</sub> (Promoter only: [[Part:BBa_K1379000|BBa_K1379000]], w/ BBa_E0240: [[Part:BBa_K1379002|BBa_K1379002]]) and P<sub>comFA</sub> (Promoter only: [[Part:BBa_K1379001|BBa_K1379001]], w/ BBa_E0240: [[Part:BBa_K1379003|BBa_K1379003]]). <i>E. coli</i> colonies holding the resulting constructs [[Part:BBa_K1379005|BBa_K1379005]] and [[Part:BBa_K1379007|BBa_K1379007]] in [[Part:pSB3K3|pSB3K3]] were observed under 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. Measurement kits for P<sub>celA</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.
 
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As shown below, only in the presence of &sigma;<sup>X</sup> would P<sub>chbB</sub> and P<sub>comFA</sub> be turned on, therefore, &sigma;<sup>X</sup> is functional.
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As shown below, only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, therefore, &sigma;<sup>X</sup> is functional.
  
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[[File:pchbBpcomFAphoto.png|800px|thumb|center|'''Figure 1. P<sub>chbB</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.''' P<sub>chbB</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter [[Part:BBa_J23101|BBa_J23101]] in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.]]
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[[File:pcelApcomFAphoto1.png|800px|thumb|center|'''Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.''' P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter [[Part:BBa_J23101|BBa_J23101]] in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.]]
  
 
Extensive documentation of our characterization can be found at our [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization characterization page for Pneumosensor].
 
Extensive documentation of our characterization can be found at our [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization characterization page for Pneumosensor].
 
<br>
 
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Please refer to the pages [[Part:BBa_K1379000|BBa_K1379000]] (P<sub>chbB</sub>) and [[Part:BBa_K1379001|BBa_K1379001]] (P<sub>comFA</sub>) for detailed characterization on the promoter activities in presence of &sigma;<sup>X</sup>.
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Please refer to the pages [[Part:BBa_K1379000|BBa_K1379000]] (P<sub>celA</sub>) and [[Part:BBa_K1379001|BBa_K1379001]] (P<sub>comFA</sub>) for detailed characterization on the promoter activities in presence of &sigma;<sup>X</sup>.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Reference===
 
===Reference===
 
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-2079
 
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-2079
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<br>
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<br><br>
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Luo Ping, & Morrison Donald. (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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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>
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<br><br>
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Piotrowski Andrew, Luo Ping, & Morrison Donald. (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.'' doi:10.1128/JB.01750-08<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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Rhodius Virgil., Segall-Shapiro Thomas., Sharon Brian., Ghodasara Amar., Orlova Ekaterina., Tabakh Hannah., . . . Voigt Christopher. (2013). ''Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.'' doi:10.1038/msb.2013.58
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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

Latest revision as of 13:44, 17 October 2014

S. pneumoniae σx CDS

Usage and Biology

σX (ComX) is an alternative σ factor in Streptococcus pneumoniae which serves as a competence-specific global transcription modulator. (BioCyc) In S. pneumoniae, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the Competence Stimulating Peptide (CSP). (Luo & Morrison, 2003) Upon stimulation by CSP, σX will be expressed and associate with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by σX to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by σX were found to contain a 8-bp consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009)

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). The principle depicted applies to σX, which when paired with the promoters it regulates, achieves orthogonal expression in E. coli. It should be noted that iGEM 2014 Hong_Kong_HKUST Team has compared the amino acid sequences of ECFs described by Rhodius et al. with that of σX from S. pneumoniae, and found no significant similarities, thus σX is a new sigma factor added to the list.

iGEM 2014 Hong_Kong_HKUST Team has cloned σX from S. pneumoniae strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters: PcelA(BBa_K1379000) and PcomFA (BBa_K1379001).

Characterization

To test the functionality of σX, we first enabled constitutive expression of σX in the σX Generator BBa_K1379006. This generator, which expresses σX, was assembled with the promoter measurement kit BBa_E0240 with either promoter PcelA (Promoter only: BBa_K1379000, w/ BBa_E0240: BBa_K1379002) and PcomFA (Promoter only: BBa_K1379001, w/ BBa_E0240: BBa_K1379003). E. coli colonies holding the resulting constructs BBa_K1379005 and BBa_K1379007 in pSB3K3 were observed under 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. Measurement kits for PcelA PcomFA without σX Generator were used as negative controls for function of σX.

As shown below, only in the presence of σX would PcelA and PcomFA be turned on, therefore, σX is functional.

Figure 1. PcelA and PcomFA promoters activated in presence of σX. PcelA and PcomFA gave little GFP signal in the absence of σX but has comparable activity as reference promoter BBa_J23101 in presence of σX. Scale bar = 5mm.

Extensive documentation of our characterization can be found at our [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization characterization page for Pneumosensor].
Please refer to the pages BBa_K1379000 (PcelA) and BBa_K1379001 (PcomFA) for detailed characterization on the promoter activities in presence of σX.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 298


Reference

BioCyc was retrieved from http://www.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