iGEM 2021: Mingdao

Mingdao

PROTOCOL

Protocol

Our project focused on phage engineering including phage isolation, plaque assay, phage purification, phage gDNA extraction, in vitro TXTL & DNA integration assay, and RCA assay. These protocols were provided as follows. Other general procedures used in gene cloning including plasmid extraction, PCR, restriction enzyme digestion, ligation and transformation, etc. as well as protein purification and analysis were mentioned in our previous iGEM projects or helped by our partner of team CSMU_Taiwan.

PHAGE ISOLATION & PLAQUE ASSAY

This protocol was provided by Prof. Chih-Hsin Hung¹ and slightly modified in our study.

↓Get the liquid sample from any environmental source like river, rain, pool, wastewater, sewage, etc.

↓Filtrate the sample with a coffee filter paper to remove the particulate in the solution.

↓Add 0.25 g of LB powder into 10 ml of the filtrate.

↓Put 10 μl (1:1000) of an overnight culture of the bacterial host (or you can try 1:100 instead)

↓Shake at 37°C for 4 hours (or 12-16 hr in some case)

↓Centrifuge at 14200 ×g for 30 min to remove bacteria or debris

↓Shake at 37°C for 4 hours (or 12-16 hr in some case)

↓Filtrate by a sterile membrane filter of 0.45 μm pore size (supposedly containing the host specific phage)

↓Prepare soft agar with 0.75% agar in LB broth autoclaved and cooled to 48°C.

↓Add 100 μl of the filtrate with phages and 100 μl of the same bacterial host into 5ml of the soft agar.

↓Overlay the soft agar with phages and bacteria onto a LB agar plate. Incubate at 37°C for several hours to 1~2 days.

↓Generally, the plaques with host-specific phages should appear in 4-24 hr.

PHAGE GENOMIC DNA EXTRACTION

With regard to the safety concerns of using phenol or chloroform and the lack of an ultracentrifuge, we’ve extracted gDNA of phages by a commercial Phage DNA Isolation Kit² (Norgen Biotek Corp.). Based on the manufacture’s instruction, the protocol was briefly described as follows.

↓Transfer 1 ml of the phage lysate into a 15-ml tube.

↓Add 500 μl of Lysis Buffer. Vortex vigorously for 10 sec.

↓Incubate at 65°C for 15 min. Inverting the tube several times during incubation.

↓Add 320 μl of isopropanol to the lysate. Mix by briefly vortexing.

↓Transfer up to 650 μl of the mixture to a spin column in the collection tube. Centrifuge at 6,000 xg for 1 min. Discard the flow through. Repeat this step until all the mixture pass through the column.

↓Add 320 μl of isopropanol to the lysate. Mix by briefly vortexing.

↓Wash the column 3 times by 400 μl of Wash Solution (containing ethanol) along with the centrifugation at 6,000 xg for 1 min.

↓Thoroughly dry the column by centrifuging at 14,000 xg for 2 min.

↓Elute phage gDNA with 75 μl of Elution Buffer. Centrifuge at 6,000 xg for 1 min.

↓The purified DNA can be stored at -20°C or analyzed by running gel electrophoresis in 1% agarose gel.

IN VITRO TRANSCRIPTION-TRANSLATION (TXTL)

Cell-free TXTL was used a lot in our studies. Go to our MEASUREMENT page to see how we established an easy-to-use and versatile TXTL system including material preparation, protocol and part characterization.

IN VITRO INTEGRATION ASSAY BY TOL2 TRANSPOSON SYSTEM

We performed the assay based on the paper of Jun Ni, et al.³

↓The blunt end KanR/Tol2, GFP/Tol2, BLUE/Tol2 fragments were generated as Tol2 mobile inserts by PCR using a high-proofreading KOD- plus DNA polymerase (TOYOBO Inc.) with forward primer Tol2-F (5′- CAGAGGTGTAAAGTACTTGA-3′), reverse primer Tol2-R (5′- CAGAGGTGTAAAAAGTACTC-3′) from templates of KanR/pTol2 (BBa_K3728004), ldhp-GFP-Tr/pTol2 (BBa_K3728005), and ldhp-amilCP- Tr/pTol2 (BBa_K3728007), respectively.

↓The PCR products were treated with DpnI to remove the template of plasmid DNAs followed by DNA cleanup with a kit (Gel/PCR DNA Fragments Kit, Geneaid Biotech Ltd.).

↓0.5 pM of the plasmid pSB1C3 (as a target), PCR products (Tol2 mobile inserts) and 27 pM His-Tol2 purified from TXTL reaction were mixed in MOPS buffer (25 mM MOPS, pH 7.0; 1 mM MnCl₂; 50 mM NaCl; 5% glycerol; 2 mM DTT; 100 ng/μl BSA)

↓Incubate at 30°C for 2 hours.

↓The resulting DNAs were cleaned up and subjected to transform E. coli DH5α competent cells.

↓Culture bacteria on LB agar plates supplemented with indicated antibiotics at a 37°C incubator.

ROLLING CIRCLE AMPLIFICATION (RCA) ASSAY

Based on the experience and suggestion of iGEM team TAS_Taipei⁴, we performed RCA assay as follows.

ssDNA Circularization

↓Set the reaction:

↓Incubate at room temperature for 4 hr

↓Add 10U of exonuclease I and 100U of exonuclease III

↓Incubate at 37°C for 1 hr

↓Inactivate enzymes at 80°C for 20 min

↓Check circular DNA formation by running gel electrophoresis in 2.5% agarose gel

RCA Assay

↓Set the reaction:

↓Incubate at 30°C for 1 hr (10 min or 40 min also tested for good results)

↓Mix 20 μl of RCA product with 1 μl of EvaGreen Dye

↓Incubate at 37°C for 1 hr

↓Transfer to a 384-well black microplate

↓Read data at Ex/Em = 500/530 nm with a microplate reader (BioTek Synergy H1)

REFERENCE

1. Prof. Chih-Hsin Hung’s lab page at Department of Chemical Engineering in I-Shou University.

2. Phage DNA Isolation Kit (Cat. 46800, 46850) webpage at Norgen Biotek Corp.

3. 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

4. Team TAS_Taipei of iGEM 2020 wiki

Team:Mingdao/Protocol