Difference between revisions of "Part:BBa K1379045"

Revision as of 20:45, 11 October 2014

S. pneumoniae σ^x-B0015

This is σ^X CDS (BBa_K1379004) followed by double terminator BBa_B0015. iGEM 2014 Hong_Kong_HKUST Team has created this part to facilitate assembly of σ^X to promoters and RBSs of choice.

Usage and Biology

σ^X (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 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)

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 σ^X, 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 σ^X from S. pneumoniae, and found no significant similarities, thus σ^X is a new sigma factor added to the list.

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).

Characterization

This construct is a sub part of the σ^X Generator BBa_K1379006 which expresses σ^X. The generator 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 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 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 retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-2079
Ping Luo., & Donald A. Morrison. (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
Andrew Piotrowski., Ping Luo., & Donald A. Morrison . (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
Virgil A Rhodius., Thomas H Segall-Shapiro., Brian D Sharon., Amar Ghodasara., Ekaterina Orlova., Hannah Tabakh., . . . Christopher A Voigt. (2013). Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. doi:10.1038/msb.2013.58