Design and Proposal of New Parts from literature
Our project involves bioremediation of antibiotics from animal excreta prior to their release into the environment or use as manure in agriculture. For this purpose we introduce a novel two component flavin dependent monooxygenase system for sulfonamide degradation. This system is naturally present in Microbacterium sp. CJ77 and was first described by Kim et al., 2019. The two component system includes the two genes sulX (sulfonamide monooxygenase) and sulR (flavin reductase). These two genes have not been reported in iGEM thus far. We propose these parts for the very first time in iGEM and have documented the literature data for these parts in the parts registry (BBa_K3519000, BBa_K3519001). We propose the use of these genes downstream to a medium strength expression system as described in the Design page (BBa_K3519006, BBa_K3519007). Theoretically, the cell lysate of E. coli expressing this system, when incubated with the animal excreta, would be able to degrade the sulfonamides in it. We have mathematically modelled these parts for their function and have predicted their structure. However, these parts are yet to be characterised in the laboratory.
To reduce the chances of horizontal gene transfer through conjugation we incorporate the genes traS and traT, (BBa_K3519002, BBa_K3519003) these parts have not been used in iGEM before and their data from literature have been added to their parts registry. These have been used downstream to the medium strength expression system as described in the design page (BBa_K3519008, BBa_K3519009). These genes are naturally found in the F factor of E. coli. The simultaneous overexpression of these genes in the absence of detergents has been shown to reduce conjugation significantly (Achtman et al., 1977). We propose that the constitutive expression of these two genes in our chassis would help in reducing the flow of DNA of our GMO into the environment. These parts have been mathematically modelled. However, these parts are yet to be studied in the laboratory.
Cloning Existing Parts
For additional safety and inducing cellular death we use the bovine pancreatic DNaseI (DNaseI) (Chen et al., 1997) downstream to the arabinose-inducible araBAD promoter (Guzman et al., 1995) (BBa_K2442101). The genetic circuit also incorporates the expression of the araC gene (BBa_K2442103). Other forms of DNaseI have been reported in iGEM. We codon optimised the sequence and optimised it for assembly compatibility and expression in E. coli (BBa_K3519004, BBa_K3519010, BBa_K3519011, BBa_K3519012). The araBAD promoter and the AraC were characterised by Team Glasgow in 2017. Only mRFP was successfully cloned downstream to the araBAD promoter. However, the clone could not be characterised for the optimum arabinose concentration due to time limitations. Following this, the DNaseI was planned to be cloned downstream to the same promoter which would have helped us measure cell death by monitoring the optical density of the medium. These experiments were done in collaboration with Team IONIS-Paris.
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
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
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
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