iGEM 2021: Mingdao

Mingdao

IMPLEMENTATION

HOW TO USE

In order to implement our project in the real world, we envision potential users to use our product as a kit consisting of (1) a vial of genetically engineered Salmonella phages with Ф29 DNA polymerase gene, (2) a homemade Luer-lock adapter with a filter-like mini Ni-NTA column, (3) a vial of the buffer containing a circular DNA template and a corresponding primer, (4) a 96-well black microplate with a lyophilized fluorescent DNA-binding dye, and (5) a wash buffer if needed. Containers, syringes, tips and a microplate reader which can measure excitation and emission wavelengths around 500 nm and 530 nm, respectively, should be prepared by the users. The detection procedure may be performed as the following flow chart.

Protocol

1. Samples to be examined including eggs and egg-related products are collected in a container (e.g., a beaker of any size, better smaller than 50ml)

2. Put liquid samples (if your sample is solid, first make it liquid by mixing with some water) into any kind of syringe or draw the samples with the syringe.

3. The engineered phage liquid in Vial A is drawn into the syringe containing the sample. Mix and incubate for 25 minutes at room temperature.

4. The Luer adapter with Ni-NTA column is locked onto the syringe. Let the mixtures pass through the column to capture any His-tagged proteins (Optional) If the sample is full of proteins or complicated contents, try to wash the column 1-3 times with the Wash Buffer in Via C we provided.

5. The reagent consisting of a circular DNA and a primer in Vial B is drew onto the column in the Luer adapter using a P200 tip. Perform RCA in the Ni column that captures His-Phi29 DNA polymerase if any. Incubate for 30 min at room temperature.

6. Push the plunger of the syringe to elute the single-stranded DNA (ssDNA) as RCA products into a well of a 96-well black microplate containing a lyophilized fluorescent DNA-binding dye such as EvaGreen® Dye.

7. Slightly shake the plate to mix the DNA and dye. Read data by measuring the signal at Ex/Em=500/530 nm. If Salmonella exists, the signal will be observed or vice versa. The limit of detection with our kit was described in detail on our page of PROOF-OF-CONCEPT.

HOMEMADE MATERIALS

◉ Ф29 Phages (Vial A)

Salmonella phages were genetically engineered to equip with phi29 DNA polymerase gene having a N-terminal His tag. The His-phi29 DNA polymerase will be produced by the phage-infected Salmonella.

◉ Ni Column (Luer-Lock Adapter)

We’ve developed a Luer-lock adapter by 3D printing, in which a mini Nickel column was embedded that was used to capture His-tagged phi29 DNA polymerase. A syringe is able to connect to one end of the adapter and a P200 tip can fit onto the other end.

◉ RCA Reagent (Vial B)

Rolling circle amplification (RCA) assay needs a circular form of single-stranded DNA and a primer that will be turn on by a phi29 DNA polymerase. The DNA polymerase was generated by the engineered phage-infected Salmonella. The other materials including the template and the primer were provided in the buffer (200 μM dNTP, 50 mM Tris-HCl, 10 mM MgCl₂, 10 mM (NH₄)₂S0₄, 4 mM DTT pH 7.5) as Vial B.

◉ Microplate (DNA Dye)

A 96-well black microplate containing lyophilized (freeze-dried) DNA binding dye (i.e., EvaGreen®) in each well was provided. The reaction will be reactivated in the elutes of RCA products.

◉ Wash Buffer (Vial C)

The Wash Buffer is composed of 20 mM imidazole, 200 mM NaCl, and 20 mM Tris at pH 8.0.

SAFETY

Our product is only used in the lab with its own regulation procedure based on government policy. There are a few or no risks associated with the application.

USERS

The potential customers were met by our team members as described in the page of Human Practices. Based on the feedback and survey, we thought egg companies and the government inspection agency could be our end users who have laboratory-based food product tests.

Egg Companies

Salmonella contamination is a leading cause in food poisoning. Egg producing and processing companies may establish a laboratory to maintain quality controls regarding food safety and shelf-life. To reduce the examination process and time, we think our product will become their first choice.

Government Inspection Agency

The government provides the Inspection Agency to ensure food safety and security. The official diagnostic methods are based on traditional culture and require further biochemistry test. The testing process is complicated and take lots of time (more than one week). We think our product will be a promising supplementary diagnostic method and help make a better decision to identify a pathogen.

PRELIMINARY RESULT

Based on our experimental data on the page of PROOF-OF-CONCEPT, we understood our Ф29 Salmonella phage as a diagnostic tool is better than traditional culture, immunoassay and PCR-based assays in terms of processing procedure and time. Regarding to genetically engineered phage, our product is comparable to the designs in the published papers (Table 1). Our Ф29 Salmonella phage can detect 20 bacterial cells/ml within 1 hour. Although the law of the Government Regulation gives zero-tolerance in food safety especially on Salmonella and defines food contamination by 1 CFU (cell) in 25 g or 25 ml of a collected sample, we still made a great achievement in the iGEM project this year.

CHALLENGES

In an interview with the chief (Dr. Huan-Lung Tao) and a technician (Ms. Wei-Xuan Zheng) of the Office of Food and Drug Safety in Taichung City, they expressed their interests in our product and are willing to test our kit. However, they also showed their concerns about the challenges they met and would come to us. More than 2000 types of Salmonella spp. may exist in the food that are necessary to be determined in an outbreak of food poisoning.

Team:Mingdao/Implementation