Difference between revisions of "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 switch point log(x₀). Figure 1 shows the measured normalized specific production rates q_P,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:

So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu et al., 2009]). The switch point of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.

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 RFU_corrected 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:

Fig. 2: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of BBa_K389016 in Escherichia coli DB3.1 in LB medium with 10 mg ml^-1 chloramphenicol and 150 µM acetosyringone.

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 q_P.

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 q_P 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 q_P 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 q_P,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.

Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], J Theor Biol 257(3):419-429.

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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