Team:Hong Kong HKUST/pneumosensor/characterization

Introduction

To test the functionality of σ^X, we first enable constitutive expression of σ^X in the σ^X Generator, BBa_K1379006. The generator was then assembled with the standard 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, which is 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 and P_comFA without σ^X Generator were used as negative controls for function of σ^X.

Results

Figure 1. P_celA and P_comFA promoters activated in presence of σ^X.

Only in the presence of σ^X would P_celA and P_comFA be turned on, as GFP expression could be seen when σ^X is present. Therefore, σ^X is functional. 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.

For characterization, P_celA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_celA Measurement Kit BBa_K1379002 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379005.

P_comFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_comFA Measurement Kit BBa_K1379003 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379007.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the P_celA and 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.

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.

References

BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867

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