We combined parts from the iGEM parts registry and sequences from Plastic Microbial Biodegradation Database (PMBD) [1] to create a construct consisting of a constitutive promoter (BBa_J23106), an RBS (BBa_B0034) and the coding sequence for BphC, an enzyme that takes part in the Biphenyl Degradation (Bph) pathway degrading diphenylmethanes, co-expressed with a his-tag. We registered this construct in the parts registry under the name BBa_K3477002 [2].
By measuring the degradation rate of the substrate, 2,3-Dihydroxy-biphenyl, the enzyme efficiency was determined and will hopefully lay the foundation for further characterization of the Bph pathway. Figure 1 shows the reaction that is performed by BphC.
Figure 1. The reaction pathway of the BphC enzyme. The 2,3-Dihydroxy-biphenyl is supposed to absorb light at 434 nm and its concentration can thus be determined through absorbance measurement. BphC enzyme can degradate 2,3-Dihydroxy-biphenyl into other substance which cannot absorb light at 434 nm. Therefore, in the measurement, we expected the absorbance will be decline by time.
Methods
An experimental design with different concentrations of substrate and enzyme was created so that the rate of degradation could be evaluated under different conditions. Parameters such as buffer content, size order of substrate and enzyme concentrations were set based on previously described methods [3]. However, the study we based our approach on did not state their enzyme concentrations. We therefore set out to vary the amount of enzyme in order to get data from both ends of the spectrum so that we could determine what amount would be the most efficient at degrading the same amount of sample. As such, 300 µl of each sample was loaded onto a nunc-96 well plate.
| 2,3-Dihydroxy-biphenyl conc. (µM) | Amount of Enzyme (µl) | Final volume (µl) | Amount of Substrate (µl) | Amount of Assay Buffer (µl) | Comment |
|---|---|---|---|---|---|
| 200 | 0 | 300 | 60 | 240 | Positive Controls |
| 400 | 0 | 300 | 120 | 180 | |
| 600 | 0 | 300 | 180 | 120 | |
| 800 | 0 | 300 | 240 | 60 | |
| 200 | 5 | 300 | 60 | 235 | Low Enzyme Concentration |
| 400 | 5 | 300 | 120 | 175 | |
| 600 | 5 | 300 | 180 | 115 | |
| 800 | 5 | 300 | 240 | 55 | |
| 200 | 10 | 300 | 60 | 230 | High Enzyme Concentration |
| 400 | 10 | 300 | 120 | 170 | |
| 600 | 10 | 300 | 180 | 115 | |
| 800 | 10 | 300 | 240 | 55 | |
| 0 | 0 | 300 | 0 | 300 | Blank |
| 0 | 10 | 300 | 0 | 290 | Alt. Blank |
The substrate, 2,3-Dihydroxy-biphenyl, has previously been shown to absorb light at 434 nm [3]. This should enable tracking of the substrate concentration over time by observing the change in intensity of absorbance at the given wavelength in a plate reader.
The correlation between absorbance and concentration is described by Beer’s law,
A = εbc
Where A is the absorbance, b is the length of the cuvette, c is the concentration and ε is the extinction coefficient. The extinction coefficient for 2,3-dihydroxy-biphenyl at pH=8 and 434 nm was previously determined to 17.9 mM-1 cm-1 [3]. Thus, the concentration can be derived directly from the absorbance. Another option to determine the concentration of a sample based on its absorbance intensity is to measure and plot a standard curve based on samples with known concentrations. The equation of a linear regression describing the correlation between the absorbance intensity and the concentration is then used to estimate the concentration of the unknown samples. In our measurement, both methods were applied when analysing the plate-reading data.
Results
The resulting absorbance intensities, as measured every 10 seconds for 8 minutes, are shown in Figure 3. Each data point is normalized to the blank (only buffer). The first noticeable feature of the data is that most data points are less than zero. This indicates that no absorbance from the substrate at the set wavelength (434 nm) can be detected, if anything the blank absorbs slightly more light than the samples with substrate. Furthermore, the sample without enzyme (used as standard) was expected to be constant at a certain intensity, but figure 3 shows fluctuations for this sample that are similar to the samples with enzyme added. Another interesting feature is that the difference between samples in which 5 µl versus 10 µl were added is non-existent, the data points for the corresponding samples were identical and can therefore not be distinguished in the plot.
Figure 2. Enzyme activity assay for BphC. The resulting intensities of absorbance at wavelength 434 nm over time for solutions with no enzyme, and enzyme with different amounts of 2,3-Dihydroxy-biphenyl (200 µM, 400 µM, 600 µM, and 800 µM). We expected the absorbance would reduce in the sample in which we added enzyme over time, but we could not see that expected result from this experiment.
No conclusions can thus be drawn about the enzyme activity from this measurement, as the substrate light absorbance doesn’t seem to occur. As a first step in the troubleshooting process of this measurement, the expected absorbance intensities according to Beer’s law (eq. 1) were calculated for each substrate concentration (eq. 2a-2d).
(1)
(2a)
(2b)
(2c)
(2d)These absorbance values were expected continuously for the No enzyme samples and as start value (Time=0) for the +enzyme samples. These results further confirm that the data achieved from the measurements presented above are inconclusive.
- [1] “PMBD - Home.” http://pmbd.genome-mining.cn/home/ (accessed Oct. 01, 2020).
- [2]“Part:BBa K3477002 - parts.igem.org.” http://parts.igem.org/Part:BBa_K3477002 (accessed Oct. 22, 2020).
- [3] L. D. Eltiss, B. Hofmann, H.-J. Hecht, H. Lunsdorf, and K. N. Timmis, THE JOURNAL OF BIOLOGICAL CHEMISTRY, " Purification and Crystallization of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase*", 1993.” https://www.uniprot.org/uniprot/P17297 (Accessed on Sep. 08, 2020).
