Difference between revisions of "Part:BBa K1379006"

Line 2: Line 2:
 
<partinfo>BBa_K1379006 short</partinfo>
 
<partinfo>BBa_K1379006 short</partinfo>
  
SigmaX Generator consisting consitutive promoter BBa_J23100, RBS BBa_B0034, ''comX'' gene, and double terminator BBa_B0015, so comX protein will be produced constitutively.
+
This is a protein generator for &sigma;<sup>X</sup> CDS ([[Part:BBa_K1379004|BBa_K1379004]]). The &sigma;<sup>X</sup> transcription is initiated by the strong constitutive promoter [[Part:BBa_J23100|BBa_J23100]] and the translation is initiated by strong RBS [[Part:BBa_B0034|BBa_B0034]], and transcription is terminated by [[Part:BBa_B0015|BBa_B0015]]
  
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
 +
&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).
 +
 +
 +
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]]).
 +
 +
 +
Following the principle depicted in Rhodius et al., () &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.
 +
 +
===Characterization===
 +
 +
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]]). E. coli 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>
 +
<br>
 +
 +
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.
 +
 +
[[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.]]
 +
 +
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>
 +
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>.
 +
<br>
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
Line 16: Line 36:
 
<partinfo>BBa_K1379006 parameters</partinfo>
 
<partinfo>BBa_K1379006 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===Reference===

Revision as of 15:15, 8 October 2014

S. pneumoniae σx Generator

This is a protein generator for σX CDS (BBa_K1379004). The σX transcription is initiated by the strong constitutive promoter BBa_J23100 and the translation is initiated by strong RBS BBa_B0034, and transcription is terminated by BBa_B0015

Usage and Biology

σX (ComX) is an alternative σ factor in Streptococcus pneumoniae which serve 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 Signal 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 (Piotrowski, Luo, & Morrison, 2009).


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: PchbB(BBa_K1379000) and PcomFA (BBa_K1379001).


Following the principle depicted in Rhodius et al., () σX 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 σX from S. pneumoniae, and found no significant similarities, thus σX is a new sigma factor added to the list.

Characterization

To test the functionality of σX, we first enabled constitutive expression of σX in the σX Generator BBa_K1379006. The generator was then assembled with the promoter measurement kit BBa_E0240 with either promoter PchbB (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 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 general negative control. for background fluorescence. Measurement kits for PchbB PcomFA without σX Generator were used as negative controls for function of σX

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

Figure 1. PchbB and PcomFA promoters activated in presence of σX. PchbB 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 (PchbB) 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
    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


Reference