Proof of Concept
INTRODUCTION
To demonstrate the potential and feasibility of our concept, we went much further than envisioning ideas and researching scientific literature to support them. Our bioreactor model passed through multiple cyles of design and analysis. To test a bench scale working our solution, we needed to design ways to check the actual degradation of halocarbons.
We started planning out assays by desiging a feasible protocol and choosing substrates. Choosing TCE as an easily available model for our assays, we began working in the lab to aim for conclusive results, that can show that the transformed bacteria shows an enhanced rate of degradation compared to the basal level. Here we present the experiments planned and carried out as a part of proof of concept.
THE DEGRADATION ASSAYS
In order to experimentally verify our hypothesis of the degradation of halocarbon refrigerants, a nonstandard experimental design must be developed. Our system is demanding in the sense that it involves switching between anaerobic and aerobic conditions along with volatile halocarbons which phase out in gaseous and liquid components. As halocarbon refrigerants are gases and difficult to handle in our available chemistry labs, we decided to start with a model substrate that resembles the halocarbon refrigerants, that would be easy to handle (liquid) and easily available. TCE (Trichloroethylene) was chosen as it has both vicinal and geminal chlorine atoms and is widely reported to undergo degradation with the enzymes of our interest using similar mechanisms as for CFCs, HCFCs and HFCs. Thus, TCE acts as a good starting point to demonstrate the degrading capability of the transformed bacteria. However, measuring the degradation rate of TCE is also a non-trivial task as it is highly volatile and must be used in extremely small amounts as TCE is toxic to Pseudomonas putida. The overall complexity of the system pushed us towards an experimental protocol that invokes the use of a gas chromatogram - as GC is very sensitive and an operationally simple method to quantify small amounts of volatile compounds. Moreover, using an internal standard like decane, we can calculate the absolute concentrations of TCE in our system.
The assay acts as a technique to verify our concept which involves degrading TCE in alternating conditions of oxygen. The assay helps us quantify the basal degradation by the bacteria and degradation of the substrate by bacteria having only Cam, only Tod, a system with Cam + Tod and compare the same between Pseudomonas putida and E. coli. The assay aids in the development of a final bioreactor model exposing the various problems that could arise at a large scale. Developments in designing the protocol for the assays were based on requirements, equipment at hand, literature survey, the dry lab model and numerous discussions with Professor Lawrence Wackett, whose inputs played a very important role in the whole of the design. Professor AT Biju and Professor Debasis Das helped us in understanding how we could use a GC for the purpose of our assays.
We first optimised the temperature settings in the GC to maximise the separation between the peaks of the solvent (Hexane, Pentane and Diethyl Ether were tried), TCE and decane. Using these optimised settings, we tried to perform the assays for Pseudomonas putida KT2440 using the protocol that can be found here.
Optimisation of GC settings
| Sr. No. | Sample | Solvent | Temp Range (°C) | Slope (°C/min) | Hold Time (min) | Observation | Remarks |
|---|---|---|---|---|---|---|---|
| 1 | Decane | Hexane | 70-300 | 20 | 0.1 | Decane peak observed (1.8 min) | - |
| 2 | TCE | Hexane | 70-300 | 20 | 0.1 | No TCE peak | - |
| 3 | TCE | Hexane | 70-180 | 5 | 0.1 | No TCE peak | Decreased Slope of Temperature change |
| 4 | - | Pentane | 70-180 | 5 | 0.1 | Pentane solvent peak for control | Pentane was chosen as it is low boiling than hexane and thus gives significant bpt difference |
| 5 | TCE | Pentane | 50-180 | 2 | 0.1 | TCE peak observed at 1.2 min | - |
| 6 | TCE | Pentane | 50-180 | 10 | 2.0 | TCE peak observed at 1.2 min | Hold time set to 2 minutes to increase delay between solvent and TCE peaks |
| 7 | TCE | Pentane | 35-180 | 10 | 2.0 | TCE peak observed at 1.6 min | Starting temperature decreased to 35 |
| 8 | TCE | Diethyl Ether | 35-180 | 10 | 2.0 | TCE peak observed at 1.6 min | Solvent switched to similar boiling Diethyl Ether as it is more easily available |
| 9 | TCE | Diethyl Ether | 35-180 | 10 | 2.0 | More intense peak on adding more TCE | Confirmation of TCE peak |
| 10 | TCE, Decane | Diethyl Ether | 30-180 | 10 | 2.0 | TCE peak observed at 1.8 min | Starting temperature decreased to 30 |
| 11 | Decane | Diethyl Ether | 30-180 | 20 | 2.0 | Decane peak observed at 5.1 min | - |
| 12 | TCE, Decane | Diethyl Ether | 30-180 | 20 | 2.0 | TCE and Decane peaks at 1.8 and 5.1 min | - |
| 13 | TCE, Decane | Diethyl Ether | 30-250 | 25 | 2.0 | Optimum Condition with both peaks well resolved | Slope increased to get faster decane peak |
TCE Degradation Assays using GC
| Sr. No. | Strain | OD | mL of TCE | mL of Et2O | Integrals of TCE peaks | Observation | |||
|---|---|---|---|---|---|---|---|---|---|
| 0 min | 20 min | 40 min | 60 min | ||||||
| 1 | KT2440 | 0.7 | 0.2 | 10.0 | - | 15215.33 | 4113.86 | 32947.31 | Peaks do not show time correlation, likely error in apparatus used |
| 2 | KT2440 | 0.7 | 0.2 | 10.0 | 6959.26 | 5978.55 | 7074.22 | 2410.22 | Peaks show consistent decline, however the 60 min point shows likely evaporation of TCE |
| 3 | KT2440 | 0.7 | 0.1 | 10.0 | - | 10217.5 | 23689.85 | 19476.44 | 20 min datapoint shows deviation |
| 4 | KT2440 | 0.7 | 0.02 | 5.0 | 8805.19 | 2643.77 | 2624.36 | 1890.83 | 0 minute was redone, others show consistent peak intensities |
| 5 | KT2440 | 2 | 0.02 | 5.0 | 2967.42 | 3061.81 | 2312.62 | 1156 | Peaks show consistent decline, however the 60 min point shows likely evaporation of TCE |
| 6 | - | - | 0.02 | 5.0 | 5416.75 | 4290.23 | 1577.61 | 2011.93 | Peaks show decline with only LB, confirming evaporation of TCE through the Teflon septum |
Summary
Of the 6 trials done, the data of the first 3 trials do not seem to follow any particular trend for us to claim that the bacteria have any basal halocarbon degradation. The randomness of the first 3 trials could be due to the fact that the supernatant was pipetted incorrectly while the sample dilution was done. Pipetting of the top layer of the supernatant after the addition of the organic solvent must be done carefully since the concentration of TCE would vary depending on the depth in which the pipette is dipped. Another reason for the randomness could be the improper stirring of the solution. The last 3 trials however show a minor decline in halocarbon degradation with time. This however could be due to dissolution and leakage of TCE through the Teflon septum or simply evaporation of the volatile substance as time passes. The whole assaying technique can be improved via pelleting the cells via centrifugation and homogenizing the supernatant. This would remove pipetting errors. Using a Teflon tape to prevent loss of halocarbons can also help improve the assays to give more accurate results.
Agilent 8890 GC at Prof. Biju's Lab
ALTERNATE WAYS TO PERFORM THE ASSAY
- Using a gas sensor-based system. Many substances like MnO2 and ZnO change their resistance due to adsorption of gases into the vacant sites of their lattice. This change in resistance can be quantified and used to measure the concentration of gases. However, on discussions with certain iGEM Teams and in general looking at papers we found lot of fluctuation in data and hence decided to discard this method of analysis.
- Headspace Analyser can be used to simply analyse the headspace gas however due to lack of equipment in nearby facilities we were unable to use this method. We also contacted Agilent technologies for this purpose but the equipment cost was way above our budget.
- Using a GC-MS, the use of a MS along with a GC gives us the molecular mass of the various quantified species along with their GC peaks. This technique is ideal to analyse the products, intermediates and substrate concentration especially if constituents are unknown. Due to lack of GC-MS facilities, we were unable to use a GC-MS for our assaying purposes and had to rely on just the GC.
- Another possible assaying technique is Ion Chromatography and Ion sequestration. In the process of degradation of halocarbons, halogen ions are released by the bacteria into the medium, whose concentrations can give an idea of the amount of substrate being degraded. This technique involves pelleting the solution after the required timestamp and analysis of the supernatant. An ion chromatography gives information on the number of different types of ions present. Sequestration of these ions selectively would give an idea of the actual concentrations of each of the ions i.e F- and Cl- ions. This technique involves a large amount of manual experimentation which is bound to cause errors and hence was not used.
PROPOSED ASSAY USING GASEOUS HALOCARBON REFRIGERANTS
- These gases being volatile would have a boiling point much lower than the solvent boiling point, causing the peaks in the GC to possibly overlap with the solvent peak, making the use of a GC for their analysis a useless effort.
- These are gases, hence a stock solution of these substances would have to be prepared unlike the case of TCE used for assaying, which is a liquid. Preparing a stock solution involves injecting a known amount of these gases into a solvent which dissolves them well, and at the same time not affecting the bacteria by being toxic to them. Methanol is found to be ideal according to Professor Wackett’s publications based on experimental trials and optimisation.
- These gases are toxic in heavy amounts and a few refrigerants are explosive as well. Hence they must be handled with care. These gases should not be allowed to leak into the atmosphere, being greenhouse and ozone depleting gases.
- So in order to use refrigerants into such an assay, a stock of solution of these refrigerants must be prepared and the analysis must be done using a Headspace gas analyser rather than using just a liquid sample in GC or GC-MS.