Part:BBa_K1379005

σ^x Generator + P_celA-E0240

ComX generator consisting constitutive promoter BBa_J23100, RBS (BBa_B0034), comX gene, and double terminator BBa_B0015 which constitutively producing comX protein, followed by ComX 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 comX protein. The comX protein will bind to 8 base pairs of PcelA (chbBp) promoter and trigger gene expression. The comX 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_J23100. 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’s working in E. coli DH10B strain or not, and to know the R.P.U (Relative Promoter Unit) with BBa_J23100 constitutive promoter as a standard constitutive promoter.

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_J23100 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 ComX 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 and Phelicase promoter induced by com-X protein drives GFP expression. PcelA and Phelicase promoter induced by com-X protein gave GFP signals. While the same construct without com-X protein did not give any GFP signals. Another negative control which is only protein comX without PcelA/Phelicase also did not give any GFP signals. Reference promoter J23100 + GFP is used as positive control. Scale bar = 5mm.

Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to J23100 constitutive promoter. PcelA promoter induced by com-X 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 J23100 promoter strength. Measurement was done by using 3 replicas.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 359
  Illegal BsaI.rc site found at 1490