In order to evaluate p-Coumaric acid production level for each construct, we used n-octanol to extract p-Coumaric acid from the bacteria growth medium. This protocol was first proposed by iGEM Uppsala in 2013, which we then optimized to suit our particular needs.
This method is based on the octanol/water partition coefficient for a specific chemical compound, which describes how a chemical compound distributed in a two-phase water/octanol system.
As the octanol/water partition coefficient remains constant under the same pH, temperature, and salinity, we conducted several experiments to see if this protocol can be optimized.
First, by using different ratios of water/octanol volume during extraction, we can obtain higher product concentration in the organic phase to increase signal intensity and thus increase the overall sensitivity of the measurement. Standard curve below was used to convert 310 nm absorbance value into p-Coumaric acid concentration in this experiment.
|500 μM p-Coumaric acid||100|
|PD2 buffer (Thermo plasmid miniprep)||100|
|PD3 buffer (Thermo plasmid miniprep)||175|
We conducted an experiment by reducing all the n-octanol volume in the tyrP-TAL functional test by one-half and one-fourth to see if there is any change in the UV-Vis absorbance profile.
We took 900 μl of wild type E. coli Nissle culture and add in 100 μl of p-Coumaric acid to make a final concentration of 50 μM. Followed by different culture/octanol ratio (1:0.05, 1:0.1, 1:0.2) of n-octanol in the final extraction step. We can amplify the 310 nm signal.
As seen in Fig. 2, higher culture/octanol ratio resulted in increase of extracted p-Coumaric acid concentration. Thus we improved the sensitivity of this protocol.
Secondly, we were able to scale down the medium usage in the extraction protocol. We conducted an experiment by reducing all the medium usage in the tyrP-TAL functional test by one-fourth and see if we are still able to construct a standard curve using this revised protocol.
We took 225 μl of E. coli Nissle culture and add in 25 μl of different concentration of p-Coumaric acid to make a final concentration of 50 μM, 25 μM, 5 μM. We then measured these samples and plotted a standard curve using these measurements. The standard curve we plotted has a satisfying R2 value. Thus, we can use this standard curve for further calibration.
|500 μM, 250 μM , 50 μM p-Coumaric acid||25|
|PD2 buffer (Thermo plasmid miniprep)||25|
|PD3 buffer (Thermo plasmid miniprep)||43.5|
This method is cheap and requires no special laboratory training. Also, the octanol/water partition coefficient is a commonly assessed parameter for a chemical compound. If your target enzymatic product is a chemical that has a positive octanol/water partition coefficient value, and no simple colorimetric reaction that can reflect its concentration, then it is highly recommended to try this n-octanol extraction method.
- 4.4: Extraction Theory. (2017, October 21). Retrieved October 20, 2019, from Chemistry LibreTexts website