Engineering Success

Even though our project faced several difficulties this year, we were successfully able to complete the first phase of The Engineering Design Cycle involving Research, Imagine and Design with certain preliminary Build. We have set up a strong foundation as a Phase I project and we expect to fulfill the remaining aspects in the Phase II of our project. During the very short period for which we had access to the laboratory, we detected and confirmed the presence of sulfonamides in poultry excreta which helped us develop our project hypothesis and design future experiments. However, in a year-wide collaboration with iGEM IONIS Paris we could successfully characterize a previously existing part and add data to it.
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Here we will look at the Research, Imagination and the Design of our project. We will also look at the proposed future experiments designed to be carried out in the Phase II of our project.

Research

Literature survey and conducting primary research is the very first step towards achieving a successful project. During the initial days of brainstorming, we came across the severity of Antimicrobial Resistance (AMR) and the steps being taken by the United Nations (UN) and WHO to combat AMR. AMR has been projected to claim 10 million deaths annually by the year 2050 [1]. On further digging we discovered the wide use of antibiotics across animal farms [2]. Here, antibiotics were not only used during infections but also as growth promoters. Furthermore, some of the antibiotics like sulfonamides have high recovery percentages in poultry excreta leading to antibiotic pollution [3]. This being an urgent issue motivated us to do further research on local poultry farms (India). Hence, we collected poultry excreta and analysed them using HPLC and MS. The results were astonishing! We successfully detected and confirmed the presence of sulfadiazine (a type of sulfonamide) in the poultry excreta. This finding led us to think and design our project hypothesis. For the proof-of-concept we chose to work with sulfonamides and we came up with a synthetic biology based solution names ‘Coli Kaze’ to combat AMR by bioremediation and processing antibiotics in the animal waste before releasing them into the environment. This way we stop the problem even before it's happening! Check out our Project Inspiration page to know more!

Imagine

Once we had the project idea in place, we wanted to incorporate different independent modules in the project that would account for the different objectives our bacteria is set to achieve. We searched for sulfonamide degrading genes that would degrade sulfonamides into simpler molecules and came across the two-component flavin dependent monooxygenase system found naturally in Microbacterium sp. CJ77 [4]. This involves the genes sulX and sulR and work together to destroy sulfonamides. To prevent our engineered bacteria (being a GMO) from undergoing Horizontal Gene Transfer (HGT) we planned to incorporate the surface exclusion genes traS and traT which help reduce conjugation significantly [5]. The death of our GMO would ensure high biosafety and to ensure that we planned to use the DNASEI gene, a highly efficient endonuclease [6], downstream to a user modulatable promoter to induce bacterial cell death and release the proteins.
Check out our Project Description page to know more!

Design

After the search for genes came the part of designing the genetic circuit. Having several teams already used the J23118 promoter for their experiments we chose to use this medium strength promoter along with the strong RBS (B0034) and call it the medium strength expression system. This system was designed to be used along with all the genes in the system except DNASEI which was designed downstream to the araBAD promoter [7][8], which is an arabinose inducible promoter. All these systems were mathematically modelled and simulated for their theoretical expression as we predicted their behaviour.
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Design of Future Experiments

Quantification of sulfonamides in animal excreta

Estimating the concentration of sulfonamides is crucial for determining the amount of enzymes required and the degradation time. This can be obtained by running the samples through the HPLC column and then comparing it with standard plots for sulfa drugs. The collected excreta samples will be oven-dried at 70℃ and then powdered. A set of samples will be prepared by dissolving known concentrations of the powdered excreta in the solvent (methanol). This gives a range of samples varying from extremely diluted to concentrated. These excreta samples will then be filtered using a PVDF filter and run through the HPLC column following the standardized method (Check out the Protocol page). The output data for different samples will be plotted as graphs and will be compared with the standard plot to get the concentration. The formula for calculating the concentration from peaks heights (PH) is as follows:
Cstandard /PHstandard = Cunknown/PHunknown
Cstandard = concentration of standard sulfa drug dissolved in methanol
Cunknown = concentration of excreta sample dissolved in methanol
PHstandard = Peak height of standard sulfa drug dissolved in methanol
PHunknown = Peak height of the excreta sample dissolved in methanol

Once the concentration for different samples is calculated, the error bar will be determined to get the range of the sulfonamides. This will be further used for sulfonamide degrading enzyme kinetic calculations.

Gene Cloning