Module 1

Antibiotic Degradation

Module 1 involves degradation of sulfonamides in the animal excreta. For this purpose the genes sulX and sulR were integrated downstream to a medium strength expression system. The genes sulX and sulR are a two component flavin dependent monooxygenase system present in Microbacterium sp. CJ77 and were first described by Kim et al., 2019. The expression of these genes in E. coli has been shown to degrade sulfonamides in the media. These parts have not been described in iGEM yet.

This part contains the gene sulX downstream to the medium promoter (BBa_J23118) and strong RBS (BBa_B0034), and will be cloned in the vector pSB1C3. sulX encodes for the flavin dependent sulfonamide monooxygenase (SulX) which helps in the degradation of sulfonamides.

This part contains the gene sulR downstream to the medium promoter (BBa_J23118) and strong RBS (BBa_B0034), and will be cloned in the vector pSB3K3. sulR encodes for flavin reductase (SulR) which helps in replenishing FMNH₂ in the media.

Module 2

Conjugation Reduction

Module 2 involves reducing conjugation thereby reducing horizontal gene transfer (HGT). To achieve this, the genes traS and traT are integrated downstream to a medium strength expression system in a F- chassis. The simultaneous expression of these genes in E. coli in a detergent free media has shown to reduce conjugation by 33,000 times (Achtman et al., 1977). The genes traS and traT help in surface exclusion and are carried naturally by the E. coli F factor. These genes have not been exploited in iGEM so far.

This part contains the gene traS downstream to the medium promoter (BBa_J23118) and strong RBS (BBa_B0034), and will be cloned in the vector pSB1C3. Overexpression of traS has been shown to prevent DNA transfer between stable mating pairs.

This part contains the gene traT downstream to the medium promoter (BBa_J23118) and strong RBS (BBa_B0034), and will be cloned in the vector pSB3K3. Overexpression of traT has been shown to prevent the formation of stable mating pairs between bacteria.

Module 3

DNA Degradation and Cell Death

Module 3 involves designing an user modulated ‘kill switch’ to induce cell death in the engineered bacteria. For this, we take use of the arabinose inducible promoter which was designed by Team Glasgow in 2017. The reason for choice of this promoter could be attributed to the high expression, tight regulation in presence of glucose and a cost effective inducer (Guzman et al., 1995). The toxin used downstream to this promoter is the bovine pancreatic DNaseI which is a well characterised endonuclease. This gene was characterised for expression in E. coli by Chen et al., 1997. The circuit incorporates the araC gene which is expressed in the opposite direction to that of the DNaseI. Both pSB3K3 and pSB1C3 will contain the kill switch to ensure biosafety.

The araC (BBa_K2442103) has been integrated downstream to the medium strength expression system. AraC is the repressor of the araBAD promoter (BBa_K2442101) and binds to the promoter in the absence of L-arabinose. L-arabinose binds to the AraC protein and removes the complex from the promoter thereby activating the araBAD promoter. Initially the mRFP (BBa_E1010) is cloned downstream to the araBAD promoter which is used to determine the optimum concentration of arabinose required for maximum induction of the promoter. Following this, mRFP is replaced by DNaseI which is used to induce cell death by DNA degradation.

References

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
https://2017.igem.org/Team:Glasgow
https://www.uniprot.org/uniprot/P00639