Difference between revisions of "Part:BBa K1379004"

Revision as of 14:49, 8 October 2014

S. pneumoniae σ^x CDS

Usage and Biology

σ^X (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). Upon stimulation by CSP, σ^X will be expressed and associate with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by σ^X to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions.

iGEM 2014 Hong_Kong_HKUST Team has cloned σ^X from S. pneumoniae strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters: P_chbB(BBa_K1379000) and P_comFA (BBa_K1379001).

Characterization

To test the functionality of σ^X, we first enabled constitutive expression of σ^X in the σ^X Generator BBa_K1379006. The generator was then assembled with the promoter measurement kit BBa_E0240 with either promoter P_chbB (Promoter only: BBa_K1379000, w/ BBa_E0240: BBa_K1379002) and P_comFA (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 P_chbB P_comFA without σ^X Generator were used as negative controls for function of σ^X

As shown below, only in the presence of σ^X would P_chbB and P_comFA be turned on, therefore, σ^X is functional.

Figure 1. P_chbB and P_comFA promoters activated in presence of σ^X. While the same construct without SigmaX protein did not give any GFP signals. Another negative control which is only protein sigmaX without PcelA or Phelicase also did not give any GFP signals. Reference promoter BBa_J23101 + GFP is used as positive control. Scale bar = 5mm.

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

@@ Line 11: / Line 11: @@
 ===Characterization===
-To test the functionality of &sigma;<sup>X</sup>, we first enable 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 standard 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.
+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>
-[[File:pchbBpcomFAphoto.png|800px|thumb|center|'''Figure 1. PcelA and 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 PcelA or Phelicase also did not give any GFP signals. Reference promoter [[Part:BBa_J23101|BBa_J23101]] + GFP is used as positive control. Scale bar = 5mm.]]
+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>.''' While the same construct without SigmaX protein did not give any GFP signals. Another negative control which is only protein sigmaX without PcelA or 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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