Team:Hong Kong HKUST/pneumosensor/characterization

Introduction

In our characterization, comX and P_celA (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. P_celA and P_helicase 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.

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 constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379002 which is PcelA promoter with GFP generator but without ComX generator.

The method of measuring RPU (Relative Promoter Unit) of P_helicase promoter is similar to measuring RPU of P_CelA 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, P_helicase, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379002 which is PcelA promoter with GFP generator but without ComX generator.