Coding
ComX

Part:BBa_K1379004

Designed by: Jordy Evan, Nadia Benedicta, Angeline Widjaja   Group: iGEM14_Hong_Kong_HKUST   (2014-09-23)
Revision as of 13:43, 8 October 2014 by Jordyevan (Talk | contribs)

S. pneumoniae σx CDS

Usage and Biology

σX is an alternative σ factor in Streptococcus pneumoniae which serve as a competence-specific global transcription modulator. ecocycsigX 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, 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.

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).

Characterization

To test the functionality of Sigma X protein, Construct consisting SigmaX generator-PcelA promoter-GFP generator (BBa_K1379005), and SigmaX generator-Phelicase promoter-GFP generator (BBa_K1379007) are built. In order to prove that SigmaX induce these two promoters, GFP expression were observed under microscope with positive control (BBa_I20260), and negative control consisting only PcelA-GFP without SigmaX generator BBa_K1379002, and Phelicase-GFP without SigmaX generator BBa_K1379003



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 BBa_J23100 + 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


[edit]
Categories
//cds/transcriptionalregulator
//chassis/prokaryote/ecoli
Parameters
None