Part:BBa_K2918054

P_BHR sp1 promoter

na

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The P_{Bhrsp1 v2} was characterized by comparing it to a T7 promoter. As a reporter, a GFP fluorescence readout was used. In order to measure fluorescence, we use a flow cytometer.
The GFP used as readout was Juniper GFP BBa_J97001 . The ribosome binding site was our Universal RBS . All of these parts were cloned into a level 1 backbone pICH47761
The protocol for preparation of samples for the flow cytometry assay is as follows:

1. Samples were grown overnight
2. Overnight cultures were diluted to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes.
3. Overnight cultures were diluted 1:100 into 5 mL, and grow for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with 1 mM IPTG where necessary
4. Samples were kept at 4 degrees for 1 hour

In the measurement, E. coli Top 10 cells without a plasmid were used as a blank. The gating for flow cytometry was determined by eye by selecting the densest region of the blank. Furthermore, the fluorescence histogram was gated to discern between cells that were 'on' and 'off', as in expressing fluorescence or not. Only cells of similar forward and side scatter were compared. The median fluorescence intensity of the blank is subtracted from the fluorescence intensity of the samples to correct for autofluorescence. In figure 1 we plot the corrected fluorescence of the samples. Figure 2 shows the gating based on size and figure 3 shows the fluorescence histogram of each sample.

Figure 1: Fluorescence corrected for autofluorescence of E. coli Top 10 cells without a plasmid.

Figure 1 shows that the strength of P_Bhr in E. coli is significantly higher than a T7 promoter induced using 1 mM IPTG for 4 hours. P_{Bhrsp1 v2} contains the binding site for TALEsp1. The presence of the binding site seems to reduce the strength of the promoter.

Figure 2: Scatter plot of forward and side scatter of E. coli Top 10 cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 1.

Figure 3: Raw fluorescence values. Black is E. coli Top 10 cells without a plasmid. Pink is T7 and blue is Pbhr_{sp1 v2}