Team:IISER-Tirupati India/Engineering

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. Aditionally, in a year-wide collaboration with iGEM IONIS Paris we could successfully develop clones with our 'kill switch' and are yet to characterise them.
Check out our Partnership page!

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.

Engineering design cycle

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.
Check out our Project Design and Model page to know more!


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

The genes sulX and sulR will be cloned into the vectors pSB1C3 and pSB3K3 respectively and will be transformed into calcium chloride competent DH5α cells individually in the presence of respective antibiotics. The colonies will be screened for positive clones, thereafter the colonies will be cultured followed by plasmid isolation. The recombinant plasmids will then be co-transformed into competent BL21 cells. Similar cloning strategy will be followed to clone traS, traT and DNaseI into the same bacterium.


Action of sulfa drugs on the engineered bacteria

The effect of different sulfa drugs on the clone and non-resistant control bacteria will be measured by broth assays. Both bacteria will be cultured in media containing different sulfonamides and the survival advantage of the clone will be measured over the control bacteria. Following this, it will be measured if sulX or sulR when present alone in the bacteria provide survival advantage and this will be quantified. To experimentally show this, individual clones expressing SulX or SulR will also be generated. Broth assays and disk-diffusion assays with different concentrations of sulfonamides will be conducted and quantified wherever possible. The experiments will be repeated at least three times and the required statistical tests would be carried out. We expect to see a significant difference between the survival of clones with both genes (containing both sulX and sulR) and clones containing either sulX or sulR.


Quantification of the rate of sulfonamide degradation

The clone will be cultured in different concentrations of sulfonamides at different environmental conditions in minimal media. This media will be collected at regular intervals and analysed by the HPLC method developed to quantify sulfonamide degradation by the clone. The methods for detecting sulfonamides have already been established as a part of preliminary feasibility study (Check out our Protocol page).


Determining the percentage reduction in conjugal frequency

The conjugal donor strain MFDpir containing the part K873003 will be mated along with our bacteria (expressing TraS and TraT, containing two fluorescence markers on either of the plasmids) in appropriate antibiotic containing media and the transconjugants will be selected based on fluorescence and donor’s antibiotic resistance marker. The Mating will be done separately for engineered bacteria and control strain. The effective colonies would be positive for both the antibiotic markers and should express both GFP and RFP simultaneously.The final reduction in conjugal transfer will be calculated using the formula below-


Reduction in conjugal transfer = (control recipients - engineered recipients)*100/control recipients


Inducible expression of bpDNaseI and its purification

DNaseI is used here as a death-inducing gene that cleaves DNA into smaller fragments thus inducing cellular death. This gene will be cloned downstream to the tightly regulated araBAD promoter into both the vectors. This will be transformed into BL21 cells. These cells will be stimulated with the appropriate arabinose concentration and cell death will be measured by monitoring the OD of the culture. The culture will be plated at regular intervals to determine the CFU. Also, DNA from such cultures will be isolated and run on an agarose gel to check for any intact DNA prevailing. The bpDNaseI will also be overexpressed and will be purified for in-vitro characterization to determine its DNA degrading efficiency.


References

1. https://www.who.int/news-room/detail/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis (retrieved on 03.10.2020)

2. Meek RW, Vyas H, Piddock LJ. Nonmedical Uses of Antibiotics: Time to Restrict Their Use? PLoS Biol. 2015 Oct 7;13(10):e1002266. doi: 10.1371/journal.pbio.1002266 . PMID: 26444324 ; PMCID: PMC4621705.

3. Martínez-Carballo E, González-Barreiro C, Scharf S, Gans O. Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria. Environ Pollut. 2007 Jul;148(2):570-9. doi: 10.1016/j.envpol.2006.11.035. Epub 2007 Feb 8. PMID: 17291647.

4. Kim DW, Thawng CN, Lee K, Wellington EMH, Cha CJ. A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria. Environ Int. 2019;127:206-215. doi:10.1016/j.envint.2019.03.046

5. Achtman M, Kennedy N, Skurray R. Cell--cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proc Natl Acad Sci U S A. 1977;74(11):5104-5108. doi:10.1073/pnas.74.11.5104

6. Chen CY, Lu SC, Liao TH. Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme [published correction appears in Gene 1998 Jun 15;213(1-2):221]. Gene. 1998;206(2):181-184. doi:10.1016/s0378-1119(97)00582-9

7. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177(14):4121-4130. doi:10.1128/jb.177.14.4121-4130.1995

8. http://2017.igem.org/Team:Glasgow