Part:BBa_K389016:Experience
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Applications of BBa_K389016
Transfer function of BBa_K389016
The data for the transfer function was measured and analyzed as described below. The data was fitted with a dose response function of the form
with the hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the inflection point log(x0). Figure 1 shows the measured normalized specific production rates qP,n plotted against the logarithm of the concentration of the inductor acetosyringone in µM. The fit has an R2 = 0.99.
The important data from the transfer function is summarized in table 1:
Table 1: Data from the transfer function for the part BBa_K389016.
| Parameter | Value |
|---|---|
| Hill coefficient | 1.673 |
| Inflection point | 26.5 µM |
| Top asymptote | 2.62 |
Data analysis for BBa_K389016
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample:
In the second step the RFUcorrected of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style:
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below:
The product accumulation in a cultivation can be described as:
with the amount of product P, the cell count X and the specific production rate qP.
RFU is commensurate to the concentration of mRFP (P) and the OD600 is commensurate to the cell count (X) ([http:/parts.igem.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]):
With these assumptions it is possible to calculate the specific production rate of mRFP qP in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD600 data:
The specific production rates qP of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system qP,0:
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) (Canton and Labno, 2004; Pasotti et al., 2009):
Protocols
Cultivation
- Inoculate 10 mL LB containing desired Antibiotic with glycerol stock
- Cultivate over night at 37 °C and 175 rpm
- Measure the OD600
- Prepare shake flasks with LB, antibiotic and different concentrations of the inductor acetosyringone
- For mRFP measurement at least 20 mL starting volume
- Inoculate the main culture with a starting OD600 of 0.1
- Cultivate at 37 °C and 175 rpm
- Take a sample at least every hour and measure the OD600
Measurement
- Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
- Freeze samples at -80 °C for storage
- To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
- Measure the fluorescence in a platereader (we used a Tecan Infinite® m200 platereader ) with following settings
- 20 sec orbital shaking (1 mm amplitude with a frequenzy of 87.6 rpm)
- Measurement mode: Top
- Excitation: 584 nm
- Emission: 620 nm
- Number of reads: 25
- Manual gain: 150
- Integration time: 20 µs
References
Canton B and Labno A (2004) Data processing of Part BBa_F2620, https://parts.igem.org/Part:BBa_F2620:Experience/Endy/Data_analysis.
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, https://parts.igem.org/Part:BBa_J23101:Experience.
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