Team:Hong Kong HKUST/pneumosensor/characterization

For characterization, P_celA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_celA Measurement Kit BBa_K1379002 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379005.

P_comFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_comFA Measurement Kit BBa_K1379003 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379007.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the P_celA and P_comFA Measurement Kits with and without the σ^X generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, BBa_I20260 was used as a positive control; BBa_E0240 was used as the negative control for background fluorescence.

Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). E. coli DH10B strains holding the constructs with or without σ^X generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter BBa_J23101 measurement device BBa_I20260 to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.

Construction

1. Construct σ^x Generator (BBa_K1379006)-P_celA-BBa_E0240-pSB3K3
Or migrate BBa_K1379005 to pSB3k3.

2. Construct σ^x Generator (BBa_K1379006)-P_comFA-BBa_E0240-pSB3K3
Or migrate BBa_K1379007 to pSB3k3.

3. Transforming P_celA-BBa_E0240-pSB3K3

4. Transforming P_comFA-BBa_E0240-pSB3K3

5. Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit

6. Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.

Measurement

1. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page)

2. Culturing E. coli DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) in falcon tubes with M9 minimal medium;

3. Take out 20-30μl of overnight cell culture with Multichannel Pipetman and mix it with M9 medium in the 96 Deep Well plate.

4. Incubate in 37°C and shake for 3 - 4 hours.

5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom.

6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5);

Filter used:
- Absorbance :Photometric 595nm,
- Excitation :485nm FITC,
- Emission :535nm FITC,
- Mirror module : FITC (403) at bottom.

- In between measurements, keep incubating the cells in 37°C while shaking.

7. 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 the fluorescence of BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); 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.

Equation of the RPU calculation is shown below:

Equipments

1. Multichannel Pipetman.
2. Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile.
3. Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.
4. Micro test plate 96 well flat bottom, made by SARSTEDT.
5. Envision Multilabel Reader.