Team:Hong Kong HKUST/pneumosensor/characterization

To measure the R.P.U (Relative Promoter Unit) of P_celA 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 P_celA 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 σ^x gene and P_celA promoter used in the construct are both cloned from Streptococcus pneumoniae NCTC 7465 strain. The experimental construct used was BBa_K1379005, which contain σ^x generator, P_celA, 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_celA promoter with GFP generator but without σ^x generator.

The method of measuring RPU (Relative Promoter Unit) of P_comFA 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_comFA and P_celA. Fluorescence and absorbance are measured in the same time period. The experimental construct used was BBa_K1379007, which contain σ^x generator, P_comFA, 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_K1379003 which is P_comFA promoter with GFP generator but without σ^x generator.

Characterization Procedure

1. Constructing :
- σ^x Generator (BBa_K1379006)-P_celA-BBa_E0240-pSB3K3
- σ^x Generator (BBa_K1379006)-P_comFA-BBa_E0240-pSB3K3
- Transforming P_celA-BBa_E0240-pSB3K3
- Transforming P_comFA-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 the whole construct listed on procedure number 1, 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;

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. GFP intensity 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 P_celA, P_comFA, and BBa_I20260 were calculated by dividing the GFP synthesis rate with 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 P_celA and P_comFA absolute promoter activity over the averaged BBa_J23101 absolute promoter activity. R.P.U value of P_celA and P_comFA reflect the maximum GFP expression in the presence of σ^x. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P_celA and P_comFA promoter in the absence of σ^x.