Difference between revisions of "Part:BBa K1379000"

Revision as of 19:10, 11 October 2014

P_celA

Usage and Biology

P_celA (or celAp) 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, which is the site where the sigma factor σ^X BBa_K1379004 (or ComX) binds to and promotes transcription initiation. During the exponential growth of Streptococcus pneumoniae, Competence Signal 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_celA alongside with other Com-Box promoters, is turned on by σ^X and drives expression of the competence protein CelA.
Voigt and his colleagues have demonstrated that orthogonal gene expression could be achieved through the use of σ^Xs, anti-σ^Xs and synthetic promoters. (Rhodius et al., 2013) iGEM 2014 Hong)_Kong_HKUST Team has tested σ^X together with promoters P_celA. Thus, could achieve orthogonal regulation if used together with other sigma factors / ECFs.

A sigmaX-inducible promoter initiating the transcription of competence CelA protein in Streptococcus pneumoniae NCTC7465 strain. PcelA is a promoter which proves to be functional in E. coli and S. pneumonia. This promoter works when induced by SigmaX protein BBa_K1379004. The sigmaX protein will bind to 8 base pairs of PcelA promoter and trigger gene expression. The sigmaX gene and PcelA promoter used in the construct are both cloned from S. pneumoniae NCTC 7465 strain. R.P.U (Relative Promoter Unit) of PcelA is measured to represent promoter strength in reference to constitutive promoter BBa_J23101. PcelA promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.

Characterization

By linking PcelA promoter with GFP generator (BBa_E0240), and SigmaX generator(BBa_K1379006), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured when the cells are in the mid-log phase. OD595 values were measured and converted to OD600 to obtain the R.P.U of the promoter.

To measure the RPU (Relative Promoter Unit) of PcelA promoter, we adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). With the use of EnVision multilabel reader from Perkin Elmer Company, it enabled us to obtain the fluorescence and absorbance of cells over time. GFP intensity and OD595 values were measured every 30 minutes after the E. coli strains are cultured to mid-log phase.

The positive control used in this characterization is BBa_I20260 which is a constitutive promoter BBa_J23101 containing GFP generator BBa_E0240, while the negative control used in this characterization is BBa_K1379002 which is PcelA promoter with GFP generator but without SigmaX generator.

The detailed description including characterization procedure and Data processing of our characterization can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].

Figure 1. PcelA 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 also did not give any GFP signals. Reference promoter BBa_J23101 + GFP is used as positive control. Scale bar = 5mm.

Figure 2. P_celA promoter Relative Promoter Unit (RPU) is measured with reference to BBa_J23101 constitutive promoter. PcelA promoter induced by SigmaX protein gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on BBa_J23101 promoter strength. Measurement was done by using 3 replicas.

Discussion

PcelA promoter is working in E. coli DH10B strain, and express GFP when induced with SigmaX protein. In comparison to the reference promoter BBa_J23101, the R.P.U (Relative Promoter Unit) of PcelA promoter is around 0.5. From Figure 2, we could tell that this promoter is specific as there is almost no GFP expression in the absence of SigmaX protein inducer. This might indicate that the 8 basepairs combox region in PcelA promoter is specific to SigmaX. However, further characterization needs to be done to evaluate crosstalks between this inducer-promoter system.

Sequence and Features

Reference

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