Difference between revisions of "Part:BBa K1379007"

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σ^x Generator + P_comFA-E0240

σ^x generator consisting constitutive promoter BBa_J23100, RBS (BBa_B0034), sigmaX gene, and double terminator BBa_B0015 which constitutively producing σ^x protein, followed by σ^x inducible promoter P_comFA encoding GFP. P_comFA is a promoter which proves to be functional in both E. coli and S. pneumonia. This promoter works when induced by σ^x protein. The σ^x protein will bind to 8 base pairs (TACGAATA) of P_comFA promoter and trigger gene expression. The σ^x gene and P_comFA used in the construct are both cloned from E. coli NCTC 7465 strain. R.P.U (Relative Promoter Unit) of P_comFA is measured to represent promoter strength in reference to constitutive promoter BBa_J23101. P_comFA promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants.

Objective

The objective is to characterize Phelicase promoter so we know whether it iss working in E. coli DH10B strain or not, and to know the R.P.U (Relative Promoter Unit) with BBa_J23101 constitutive promoter as a reference.

Method

By linking Phelicase promoter with GFP generator (BBa_E0240), 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.

Characterization

To measure the RPU (Relative Promoter Unit) of Phelicase 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_K1379003 which is Phelicase 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. Phelicase 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 Phelicase also did not give any GFP signals. Reference promoter BBa_J23101 + GFP is used as positive control. Scale bar = 5mm.

Figure 2. Phelicase promoter Relative Promoter Unit (RPU) is measured with reference to BBa_J23101 constitutive promoter. Phelicase 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

Phelicase 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 1.2, which is higher than the reference. 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 Phelicase promoter is specific to SigmaX. However, further characterization needs to be done to evaluate the crosstalk between this inducer-promoter system.

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

Sequence and Features