Validation and testing of all aspects of a design can be done through experimentation. We have brainstormed several experiments after thorough research and questioning, keeping in mind all the risks that could be involved but due to lack of access to laboratory facilities, we were unable to actually perform and troubleshoot them.
We have divided our experiments into three types: Chassis, Composite Bio-Brick 1 and Composite Bio-Brick 2 experiments. The efficiency, relevance and safety aspects of each gene and the overall project must be tested using these.
Experiments to find the threshold concentration of methylmercury required by the transformed bacteria to generate a response and a test to analyse the tolerance of the bacteria to high levels of methylmercury chloride, pH and paraquat (oxidative stress) have been designed as part of the chassis and preliminary experiments. Further experiments have been broken down to the study of the two Bio-Bricks we have designed. Characterization of the different parts of the plasmids, determination of the most efficient combination of parts, Understanding the amount of stress required to activate the parts, showing the efficiency of the system and recognition of the compatibility and working of the two plasmids are some of the several aspects and considerations we have looked into. All of the experiments were designed without disregarding any safety concerns and keeping in mind good lab practices. To replicate the gut environment the experiments should be performed at a pH of 6-7 and 37° celsius and circuits assembled using Gibson assembly. All the measurement equipment used should be calibrated before each experiment.
Even though the methylmercury concentration that harms humans differs in different individuals, for experimental purposes we have referred to literature and chosen concentrations with known physiological effects on mice and humans Ashner M., 2012.
We have proposed Escherichia coli Nissle 1917 as our chassis. It is a known probiotic non-pathogenic strain. We have designed experiments to show that the strain used for the probiotic to be made, Escherichia coli Nissle 1917 can survive under oxidative stress, varying pH and is safe iGEM Uppsala, 2013 iGEM Tuebingen, 2019.
Figure 1: Escherichia coli Nissle 1917
Composite Bio-Brick 1
Figure 2: Composite Bio-Brick 1 - working together!
Escherichia coli DH5α inoculated with Composite Bio-Brick 1 should be induced with methylmercuric chloride and the fluorescence of the GFP must be measured on a spectrofluorometer (excitation-350nm, emission-500nm wavelength) after calibration. Measurement of O.D and GFP intensity should be done at intervals of 60 minutes for 4.5 hours after which minimum and maximum accumulation of mercury inside the cell must be estimated using gas chromatography (Detailed protocol is given in the handbooks in the download section). In this preliminary experiment one would obtain the threshold response - which can be used as the minimum limit, and the limit of tolerance of the bacteria - to be used as the maximum limit for other experiments.
Figure 3: Control mechanism for Bio-Brick 1.
We propose to use the MerR-PmerT system as the control mechanism for the project Brown et al., 2003. Response of MerR to methylmercury should be tested using 2 control circuits which do not have the MerR gene present in them. E. coli cells inoculated with methylmercury chloride are to be grown for the required amount of time according to the results of the preliminary experiment. GFP intensities must then be checked where we expect an exponential increase in fluorescence with an increase in methylmercury concentration for the circuit with MerR present. For the control with PmerT, we expect that the transcription would be initiated but at a lower level than the test circuit. We hypothesize that the plot of fluorescence and methylmercury concentration would indicate that MerR contributes significantly in methylmercury response.
Figure 4: Transport of Methylmercury
To determine the final transport design, we propose three circuits consisting of a combination of genes among MerP, MerC, MerT and MerE which should be tested. Through literature survey we have found out that three circuit designs are possible Sone Y., et al., 2013. The circuit showing the most effective results must be chosen. If the circuit with smaller length shows similar results as the main circuit , it must be chosen as it will result in reduced metabolic burden during implementation (i.e. scale-up).
Circuits we test for the final transport design system:
- MerP-MerT -MerE
To test the efficiency and characterize each of the 4 parts separately we have designed experiments with each of the parts making use of 2 test circuits and 2 controls. This should enable us to map the contribution of each gene to the overall system.
- The final transport design system
- Constitutive Promoter – RBS – (The part to be tested, i.e. MerP, MerC, MerT or MerE) – RBS – Double Terminator
- Final circuit design without the part to be tested
- Wild type Escherichia coli DH5α
Escherichia coli cells inoculated with methylmercury chloride should be grown for the required amount of time for the 2 circuits to be tested and 2 controls. After centrifugation of the cell suspension, the mercury concentration in the supernatant for each set must be mapped with gas chromatography. Plots of concentration vs time for each of the sets must be used to analyze the efficiency of the parts in transporting methylmercury.
Enzymes and working
Figure 5: Enzymes Illustration
We propose to use alkylmercury lyase and mercuric reductase to convert methylmercury to elemental mercury Mathema et al., 2011 Parks et al., 2009. The effects of methylmercury concentrations on the bacterial resistance in the presence and absence of MerA and MerB with 3 circuits must be checked - The first with presence of both MerA and MerB, the second with deletion of either MerA or MerB respectively and the third with absence of both MerA and MerB. The increase in the Mer spectrum with the introduction of MerB and MerA should be checked with the expected conclusion being that the addition of the two genes confer to a better resistance to methylmercury iGEM UFAM-UAE, 2016. MTT assay must be performed to quantify the resistance provided by each gene MerA and MerB. MTT assay must be performed to quantify the resistance provided by each gene MerA and MerB. An experiment to determine the activity of MerA and MerB must be done using which the optimal CFU for the optimal enzyme concentration can be mapped. Using inputs from MATLAB models, the enzyme concentration to be used has been determined to be in the order of 10-4 (Refer to iGEM MIT_MAHE Model Handbook for more information).
Overall working of Composite Bio-Brick 1 must be tested to show its efficiency in converting methylmercury to elemental mercury as compared to natural systems.
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Composite Bio-Brick 2
Figure 6: Composite Bio-Brick 2 - working together!
Figure 7: Control mechanism for Bio-Brick 2.
The SoxS-SoxR system is proposed as the control mechanism for Composite Bio-Brick 2 Demple, Hidalgo & Leautaud, 1998. To create an environment of oxidative stress, transformed Escherichia coli Nissle 1917 must be induced with methyl viologen dichloride (paraquat).
We test for the efficiency of the control system consisting of HlyB, HlyD, TolC and HlyA against a control with HlyB, HlyD and TolC absent. We analyse the HlyA + GFP transported outside the cells, HlyA + GFP remaining inside the cells and the total HlyA + GFP produced with absorbance vs time graphs to show the efficiency in getting the anti-inflammatory cytokines outside the cell to the target site.
The relationship between the amount of oxidative stress and transcription rates of the genes must then be measured by mapping GFP intensity after calibration. The intensity of GFP is expected to increase with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter would increase and then stabilize to a constant rate. It is also expected that there would be a higher transcription rate of SoxS promoter as the concentration of the inducer increases.
The threshold of oxidative stress required to activate SoxR and in turn activate SoxS promoter must be checked with expected result being that the activation occurs even at low oxidative stress, preventing any unwanted inflammation due to our probiotic by effective release of anti-inflammatory substances.
Transport and working
Figure 8: Transport of IL-10 fused with HlyA.
The HlyA transport system is proposed as the mechanism of transport of IL-10 outside the cell Gentschev et al., 2002. Efficiency of the transport system consisting of HlyB, HlyD, TolC and HlyA should be checked by comparison against a control with HlyB, HlyD and TolC absent. Analysis of the HlyA + GFP transported outside the cells, HlyA + GFP remaining inside the cells and the total HlyA + GFP produced with absorbance vs time graphs must be done to show the efficiency of the system to transport the IL-10 outside the cell.
Finally, the overall working of composite Bio-Brick 2 must be tested to show that it will work only in the conditions of methylmercury induced inflammation.
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Keeping in mind the importance of being able to efficiently produce the bacteria on a larger scale, we must test out factors that play an important role in the effective growth of the microbe. Inoculum properties and media components are the major contributing factors to attain optimal growth.
The main objective of inoculum development for unicellular bacterial fermentations is to produce an active inoculum, which will give as short a lag phase as possible in subsequent cultures. A long lag phase is disadvantageous in that not only is time wasted but also medium constituents are consumed in maintaining a viable culture prior to growth. The length of the lag phase is affected by the size of the inoculum and its physiological condition with mid-exponential cultures tending to reduce the length of the lag phase Stanbury et al., 2017.
We have premeditated experiments which should help in the determination of the inoculum percentage, inoculum time and inoculum age which would enable us to obtain an optimal growth curve for our process. We must also test the effects of pH and temperature on the inoculum. In addition we must calculate the doubling time of our organism in order to determine the fermentation time in the bioreactor.
Figure 9: Age of culture
Preserving the plasmid
Low-copy-number plasmids risk plasmid loss during cell division. This will lead to our probiotic to lose its therapeutic ability. Hence, we must calculate the plasmid loss rate so that we can modify the manufacturing process to optimize the generation time for the therapeutic effect.
Terrific broth, an enriched media is proposed as a suitable media for cell culture. The media contains glycerol which will be used as the main carbon source. Its composition is given below:
|Yeast Extract||24 g|
|Potassium phosphate (0.17 M monobasic, 0.72 M dibasic)||100 ml|
|Bacto Agar (Optional)||15 g|
However, we should test different media compositions to determine the most efficient media formulation.
The nitrogen content in the different formulations must be quantified using spectrophotometric methods or Kjeldahls method enabling the mapping of the nitrogen requirements and availability.
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