Difference between revisions of "Part:BBa K1379005"

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σ^x Generator + P_celA-E0240

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

Objective

The objective is to characterize PcelA promoter so we know whether it is 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 PcelA 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 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. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to BBa_J23100 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.

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