Difference between revisions of "Part:BBa K1379006"

Revision as of 13:35, 10 October 2014

S. pneumoniae σ^x Generator

This is a protein generator for σ^X CDS (BBa_K1379004). The σ^X 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

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). (Luo & Morrison, 2003) 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. Characterized genes regulated by σ^X were found to contain a 8-bp consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009)

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_celA(BBa_K1379000) and P_comFA (BBa_K1379001).

Following the principle depicted in Rhodius et al., (Rhodius et al., 2013) σ^X 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 σ^X from S. pneumoniae, and found no significant similarities, thus σ^X is a new sigma factor added to the list.

Characterization

This generator, which expresses σ^X, was assembled with the promoter measurement kit BBa_E0240 with either promoter P_celA (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 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 general negative control. for background fluorescence. Measurement kits for P_celA 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_celA and P_comFA be turned on, therefore, σ^X is functional.

Figure 1. P_celA and P_comFA promoters activated in presence of σ^X. P_celA and P_comFA gave little GFP signal in the absence of σ^X but has comparable activity as reference promoter BBa_J23101 in presence of σ^X. 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 (P_celA) and BBa_K1379001 (P_comFA) for detailed characterization on the promoter activities in presence of σ^X.
Sequence and Features

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