Difference between revisions of "Part:BBa K1379007"

Latest revision as of 13:47, 17 October 2014

σ^x Generator + P_comFA-E0240
This is P_comFA Measurement Kit BBa_K1379003 assembled with σ^X generator BBa_K1379006.

Usage and Biology

P_comFA (or comFAp) is a σ^X regulated promoter from Streptococcus pneumoniae. It is a member of the Cin-Box or Com-Box promoter family that share the 8 base pair consensus sequence of TACGAATA, where the sigma factor σ^X BBa_K1379004 (or ComX) binds to and promotes transcription initiation. (Piotrowski, Luo, & Morrison, 2009) During the exponential growth of S. pneumoniae, Competence Stimulating Peptide (CSP) mediated quorum sensing induces expression of σ^X. σ^X as a global regulator then directs S. pneumoniae to enter a transient competent cell state. P_comFA, alongside with other Com-Box promoters, is turned on by σ^X and drives expression of the competence protein comFA. (BioCyc)

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). This principle has been demonstrated to work in E. coli (see characterization below), in which P_comFA can only be turned on in the presence of σ^X.

iGEM 2014 Hong_Kong_HKUST Team has cloned P_comFA from S. pneumoniae strain NCTC7465 and characterized its Relative Promoter Units (RPU) in presence and absence of a σ^X generator BBa_K1379006. Promoter P_celA (BBa_K1379000), which is another Com-Box promoter recognized by σ^X, has also been characterized.

The transcription start site of this promoter has yet to be experimentally located.

Characterization

For characterization, BBa_K1379007 which contain P_comFA Measurement Kit BBa_K1379003 assembled with σ^X generator BBa_K1379006 in plasmid pSB3K3 was used.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the P_comFA Measurement Kits with and without the σ^X generator under a 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.

Figure 1. P_comFA is on in presence but not absence of σ^X. P_comFA Measurement Kit gave strong GFP signals comparable to BBa_I20260 when assembled without σ^X generator, and fluorescence was not observed with the same construct without the σ^X generator. Scale bar = 5mm.

Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). E. coli DH10B strains holding the constructs with or without σ^X generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter BBa_J23101 measurement device BBa_I20260 to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.

Figure 2. P_comFA has 1.21 RPU when paired with σ^X generator. P_comFA was measured in reference to BBa_J23101 constitutive promoter with and without σ^X generator BBa_K1379006. RPU shown was calculated from 3 replicas.

The above results showed that P_comFA is functional in E. coli and is turned on specifically in presence of σ^X. Further characterization can be done to evaluate crosstalk between this sigma factor-promoter pair and their other counterparts.

Extensive documentation of this characterization, including the wet lab protocols and data processing, can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page]. Sequence and Features

Reference

BioCyc was retrieved from http://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

J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3, 4. doi: 10.1186/1754-1611-3-4