PROOF OF CONCEPT
Salmonella is one of the most prevalent causes of food poisoning. Salmonellosis causes an estimated millions of illness and thousands of deaths annually worldwide. Salmonella is ranked among top 3 in bacterial foodborne infection in Taiwan1. People with Salmonella infection may develop diarrhea, fever, abdominal cramps and vomiting within 8 to 72 hours after contaminated meals or water were taken.
A bowl of instant noodles with half a boiled egg is one of the favorite and enjoyable late-night suppers for high school and college students when working hard at midnight (just like on iGEM project, for example) in Taiwan. Therefore, for food safety and security, we want to research Salmonella detection methods and develop new tools to reduce the Salmonella contamination in food chain especially in egg-related products2
Global food pathogen testing market size has been estimated to 5 billion by 2025. Bacterial infection is focused on E. coli, Salmonella and Listeria detection. The table below represents the traditional and developing methods of Salmonella diagnosis after our paper research3 and a workshop with Dr. Bowen Li of CREATIVE LIFE SCIENCE CO., LTD in Taiwan. The current methods all need initial bacterial growth or amplification and take 2-7 days based on the materials and techniques. None of them can afford the emergency testing during the pandemic. We would like to develop a novel Salmonella detection tool with synthetic biology to get the results within hours rather than days.
A bacteriophage (a.k.a phage) is a virus that infects and replicates within specific bacteria. Such a specificity is used to detect a unique type of bacteria (or narrow or short ranges of bacterial types). The technique is well known as phage typing4,5. However, phage typing relies on the plaque formed on the lawn of bacterial culture, that is, bacterial growth is required as the current Salmonella test.
Phage typing has been improved by introducing reporter genes into the engineered phage genome6,7,8. Table 2 showed the reporter phages applied in Salmonella testing. Compared to culture, immune and nucleic acid-based methods (Table 1), the response time of phage-based detection assay was reduced to hours or even minutes. However, the limitation of sensitivity (detection limit) is still to be overcome because the Salmonella contamination is zero tolerant for food safety. (i.e., Salmonella spp. should not be detected a cell in 25 g (or 25 ml) of samples under Taiwan’s government regulation law.)
Team TAS_Taipei of iGEM 2020 was inspiring us with rolling circle amplification (RCA) to detect viral targets9. RCA is a simple and efficient isothermal amplification method that utilizes phi29 DNA polymerase to generate long single stranded DNAs with a primer targeting on a circular DNA template. The amplified ssDNA can be easily measured by a fluorescent DNA-binding dye in a short time.
We are curious about what if designer phages carry phi29 DNA polymerase gene other than common GFP or Luciferase reporter genes. Are the signals being amplified stronger and more efficiently?
• Bacteriophage hunting: a phage specifically infects Salmonella
• Salmonella phage genome extraction & packaging
• Salmonella phage genome engineering
• Salmonella detection testing
deSALMONEtorTM is a Salmonella specific reporter phage designed by MINGDAO iGEM team to carry Bacillus subtilis phage phi29 DNA polymerase gene (CARGO). The phage genome will be engineered by in vitro transposon-mediated recombination through Tol2 transposon system including a Tol2 transposase (HELPER) and a Tol2 mobile element (DONOR)10. In addition, we will build up a phage genome engineering toolkit with TXTL (in vitro transcription-translation) cell-free system to synthesize and package the phage genomes11,12,13. In order to achieve engineering success, we will construct BioBrick parts such as BBa_K3728008 for Ф29 DNA polymerase gene, BBa_K3728000 for Tol2 transposase and BBa_K3728002 for Tol2 mobile element.
We anticipate we will complete the BioBrick part constructions. Tol2 transposon system will be characterized in plasmid integration assay and phage engineering. And we’ll build a TXTL system and set up standard protocols to benefit future iGEM teams and projects. The TXTL system will be tested in in vitro reporter expression assays, protein expression and purification, as well as phage genome synthesis and packaging. The Salmonella specific phages will be isolated. The phage genome will be extracted, engineered and packaged. The reporter phages with phi29 DNA polymerase will be created. And in the end, the sensitivity and efficiency of our deSALMONEtorTM reporter phage to detect Salmonella will be understood.
1. Chang YJ, Chen MC, Feng Y, Su LH, Li HC, Yang HP, Yu MJ, Chen CL, Chiu CH. Highly antimicrobial-resistant Nontyphoidal Salmonella from retail meats and clinical impact in children, Taiwan. Pediatr Neonatol. 2020 Aug;61(4):432-438. doi: 10.1016/j.pedneo.2020.03.017.
2. Whiley H, Ross K. Salmonella and eggs: from production to plate. Int J Environ Res Public Health. 2015 Feb 26;12(3):2543-56. doi: 10.3390/ijerph120302543.
3. Ricke SC, Kim SA, Shi Z, Park SH. Molecular-based identification and detection of Salmonella in food production systems: current perspectives. J Appl Microbiol. 2018 Aug;125(2):313-327. doi: 10.1111/jam.13888.
4. Rabsch W. Salmonella typhimurium phage typing for pathogens. Methods Mol Biol. 2007;394:177-211. doi: 10.1007/978-1-59745-512-1_10.
5. Wei S, Chelliah R, Rubab M, Oh DH, Uddin MJ, Ahn J. Bacteriophages as Potential Tools for Detection and Control of Salmonella spp. in Food Systems. Microorganisms. 2019 Nov 17;7(11):570. doi: 10.3390/microorganisms7110570.
6. Smartt AE, Ripp S. Bacteriophage reporter technology for sensing and detecting microbial targets. Anal Bioanal Chem. 2011 May;400(4):991-1007. doi: 10.1007/s00216-010-4561-3
7. Vinay M, Franche N, Grégori G, Fantino JR, Pouillot F, Ansaldi M. Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium. PLoS One. 2015 Jul 17;10(7):e0131466. doi: 10.1371/journal.pone.0131466.
8. Kim S, Kim M, Ryu S. Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium. Anal Chem. 2014 Jun 17;86(12):5858-64. doi: 10.1021/ac500645c.
9. Team TAS_Taipei of iGEM 2020 wiki
10. Ni J, Wangensteen KJ, Nelsen D, Balciunas D, Skuster KJ, Urban MD, Ekker SC. Active recombinant Tol2 transposase for gene transfer and gene discovery applications. Mob DNA. 2016 Mar 31;7:6. doi: 10.1186/s13100-016-0062-z
11. Tinafar A, Jaenes K, Pardee K. Synthetic Biology Goes Cell-Free. BMC Biol. 2019 Aug 8;17(1):64. doi: 10.1186/s12915-019-0685-x.
12. Shin J, Jardine P, Noireaux V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol. 2012 Sep 21;1(9):408-13. doi: 10.1021/sb300049p.
13. Rustad M, Eastlund A, Jardine P, Noireaux V. Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synth Biol (Oxf). 2018 Jan 22;3(1):ysy002. doi: 10.1093/synbio/ysy002.