Experiments
HPLC/MS
Animal excreta contributes to antibiotic pollution which in turn leads to antimicrobial resistance. Our project focuses on the removal of excessive antibiotics present in the Animal waste before releasing them into the environment. Initial literature study, helped us identify the use of antibiotics as growth promoters in poultry, and their large recovery percentages in excreta. So for proof of concept study, we decided to target sulfonamides and degrade them using our genetically engineered bacteria Coli Kaze. Confirming the presence of antibiotics in animal excreta and estimating their concentration was essential for designing our project as there was little to no data available on poultry farms from India.
Initially, we began by enquiring about the common antibiotics used in the farms from local pharmacy stores which suggested the use of sulfonamides and β-lactams. We then went on to collect the excreta samples from farms near our institute which processed on-site with 100% Methanol to neutralise bacterial or viral pathogens. Next, standard samples of sulfadiazine and ampicillin were run in HPLC to standardise the solvents and protocols. The collected samples were then run on HPLC after some preliminary treatment, which was done following the biosafety measures. Further, the mass spectrometric analysis for a collected peak confirmed the presence of sulfonamide (sulfadiazine). Unfortunately, due to time constraints we could not confirm the presence of any β-lactam in the poultry excreta.
Click me for the detailed Protocol.
Click me for the Results.
Engineering the ‘kill switch’
For proof-of-concept work, we partnered with Team iGEM IONIS Paris and with their help conducted a few experiments to show the working of ‘kill switch’. Our team did not have access to the laboratory and all experiments were conducted by our partners while we could only monitor the experiments remotely. Initially, we wanted to characterise the araBAD promoter which has already been previously reported. This would involve cloning mRFP downstream to the araBAD promoter. The characterisation would involve determining the optimum arabinose concentration required for maximum induction of the promoter (maximum fluorescence). Later, the DNASEI gene would be cloned downstream to the araBAD promoter and stimulated with the determined optimum concentration of arabinose to induce cell death.
We began by amplifying the mRFP, DNASEI and the araBAD promoter system which were ordered as gBlocks from IDT. The biobrick forward and biobrick reverse primers were used to amplify the gBlocks. At the same time, the vector backbones (pSB1A3, pSB4A5 and pSB4K5) were also amplified using the suffix forward and the prefix reverse primers. All these products were PCR purified. The mRFP/DNASEI was restriction digested with XbaI and PstI, the araBAD was restriction digested with EcoRI and SpeI, and the vector backbone was digested with EcoRI and PstI. All these digested products were then ligated simultaneously in a single pot reaction where the insert:vector ratio was maintained to be 3:1. The ligation product was directly transformed into competent E. coli and were plated overnight in antibiotic containing agar plates. The colonies from these plates were screened using colony PCR for positive clones and were repatched into another plate. The positive clones from the patched plates were used for recombinant plasmid isolation and further experiments.
Although Team iGEM IONIS could help us successfully get the clones, unfortunately, we could not do any further experiments with the clones due to time-limitation. We would like to characterise these clones in the second phase of our project where we show the working of the ‘kill switch’. We also plan to isolate the bpDNaseI enzyme and characterise its in-vitro function. Alongside the ‘kill switch’ we also plan to show the working of our other proposed new parts and characterise them. The plan is to show the working of a prototype bacteria, Coli Kaze with improved biosafety features, that is able to degrade the sulfonamides in animal waste.
