Team:Hong Kong HKUST/pneumosensor/characterization

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Pneumosensor Characterization

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ComX (BBa_K1379004)

Introduction

In our characterization, comX and PcelA (100 base pairs combox promoter) was assembled with GFP reporter (BBa_E0240) using RFC10 format (BBa_K1379005). The construct BBa_K1379005 was then transformed into DH10B cells. To provide a positive control, BBa_I20260 containing strong constitutive promoter, strong RBS, and GFP generator was transformed into DH10B cells. Our negative control, BBa_K1379002, was the same as our experimental construct, but without the comX gene.

Results

Figure 1. PcelA and Phelicase promoter induced by ComX protein drives GFP expression.
PcelA and Phelicase promoter induced by ComX protein gave GFP signals. While the same construct without comX did not give any GFP signals. Another negative control which is only GFP generator without comX and PcelA/Phelicase also did not give any GFP signals. Reference promoter J23100 + GFP is used as positive control. Scale bar = 5mm.

PcelA (BBa_K1379000)

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. The experimental construct used was BBa_K1379005, which contain comX protein generator, PcelA, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a GFP generator, while the negative control used in this characterization is BBa_K1379002 which is PcelA with GFP and without comX.

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.

Phelicase(BBa_K1379001)

Figure 3. Phelicase promoter Relative Promoter Unit (RPU) is measured with reference to J23100 constitutive promoter.
Phelicase promoter induced by ComX 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.

The method of measuring RPU (Relative Promoter Unit) of Phelicase promoter is similar to measuring RPU of PCelA which 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). It is done by using EnVision multilabel reader from Perkin Elmer Company to obtain the fluorescence and absorbance of cells over time. The experimental construct used was BBa_K1379007, which contain comX protein generator, Phelicase, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a GFP generator, while the negative control used in this characterization is BBa_K1379003 which is Phelicase with GFP and without comX.

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