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 R.P.U (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). By linking PcelA promoter with GFP generator (BBa_E0240), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured using EnVision multilabel reader from Perkin Elmer Company, 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. The comX gene and PcelA promoter used in the construct are both cloned from E. coli NCTC 7465 strain. 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.

Characterization Procedure

1. Constructing BBa_K1379005-pSB3K3 (ComX Generator (BBa_K1379006)-PcelA-BBa_E0240-pSB3K3); Transforming BBa_K1379002-pSB3K3 (PcelA-BBa_E0240-pSB3K3); Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit; Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.
2. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page)
3. Culturing E. coli DH10B strain carrying BBa_K1379005-pSB3K3, BBa_K1379002-pSB3K3, BBa_I20260-pSB3K3, BBa_E0240-pSB3K3 in supplemented M9 medium and measuring the respective growth curve;
4. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase;
5. Calculating the Relative Promoter Units (RPU) using the obtained data;

Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to J23100 constitutive promoter.

PcelA 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.

Data Processing

1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min);
2. For GFP intensity, GFP expression of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 are subtracted with the background fluorescence which is BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted; OD595 was converted to OD600, and average values were taken;
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve;
4. Absolute promoter activity of PcelA and I20260 were calculated by dividing the GFP synthesis rate of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 over the average OD600 value;
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values;
6. Finally, R.P.U was calculated by dividing the averaged PcelA absolute promoter activity over the averaged I20260 absolute promoter activity. R.P.U value of BBa_K1379005-pSB3K3 reflect the maximum GFP expression of PcelA promoter in the presence of ComX protein. Leakage could be analyzed according to the R.P.U value of BBa_K1379002-PSB3K3 which shows the GFP expression of PcelA promoter without ComX protein.

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). There are no difference in measurement condition of measuring P_helicase and P_CelA. Fluorescence and absorbance are measured in the same time period. 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 P_helicase promoter with GFP generator but without ComX generator.