Team:Calgary/Experiments/imposter

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Our Experiments

General Protocols

Rehydration of Registry DNA

Experimental Details and Rationale	Registry DNA was rehydrated for completion of the Interlab Study. Also, Part BBa_K934001 (phaC1-A-B1) was rehydrated by the Secretion subgroup and transformed into our chassis so that P(3HB) was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning.
Materials	iGEM 2017 distribution kit ddH₂O
Protocol	Add 10 μL of ddH₂O to the desired well. Pipette up and down 3-5 times. Incubate at room temperature for 10 minutes. Transform cells with 1 μL of rehydrated DNA. Store the remaining amount at -20°C.

Rehydration of IDT Synthesized DNA

Experimental Details and Rationale	Our genetic parts were ordered from IDT and arrived as a dry, lyophilized powder. They were resuspended in aqueous solution for cloning into pSB1c3 or pET29B vectors and to ligate multiple parts together.
Materials	Synthesized DNA from IDT (gBlocks) ddH₂O
Protocol	Centrifuge tube containing the synthesized DNA for 3-5 seconds at 3000g to ensure that all material is at the bottom of the tube. Add ddH₂O to reach a final concentration of 50 ng/μL. Vortex. Incubate at 50°C for 20 minutes. Briefly vortex and centrifuge . Store at -80°C.

Plasmid MiniPrep from Escherichia coli
Experimental Details and Rationale	E. coli DH5ɑ and BL21 were lysed and the pSB1c3 or pET29B vectors were isolated to be used in cloning our genetic constructs. Bacterial clones were lysed for analysis (eg: confirmation restriction digest, genetic sequencing).
Materials	2.5 mL overnight culture of bacteria in Luria-Bertani broth with appropriate buffer in 16x125mm culture tube Resuspension buffer (stored at 4°C): 50 mM Tris-HCl, pH 8 10 mM EDTA 100 μg/mL RNase A Lysis buffer: 200 mM NaOH 1% (v/v) SDS Precipitation buffer: 3 M CH₃CO₂K, pH 5.5 Isopropanol 70% ethanol Table-top centrifuge Ice bucket 2-mL microcentrifuge tubes 1.5-mL microcentrifuge tubes ddH₂O 3
Protocol	Transfer 2 mL of the overnight culture to a 2-mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant. Resuspend pellet in 300 μL Resuspension buffer. Add 300 μL Lysis buffer. Invert gently and incubate at room temperature for 3-5 minutes. Add 300 μL Precipitation buffer. Invert gently. A white precipitate should form. Centrifuge at 14,000g for 10 minutes at room temperature. Retain supernatant in a clean 1.5-mL microcentrifuge tube. Add 650 μL isopropanol. Gently invert and incubate at room temperature for 10 minutes. Centrifuge at 14,000g for 10 minutes at 4°C. Discard supernatant. Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube. Do not resuspend. Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant. Dry pellet in speed vac for 15-30 minutes, or until no more liquid remains visible in the tube. Resuspend pellet in ddH₂O and store at - 20°C.

Restriction Digest
Experimental Details and Rationale	Our genetic parts and vectors were digested with restriction enzymes before they were ligated. Plasmids isolated from transformants (through Plasmid Miniprep) were also digested for confirmation of ligation and transformation.
Materials	DNA (eg: from plasmid miniprep) Restriction enzymes 10X appropriate buffer ddH₂O 100X Bovine Serum Albumin (BSA) (if using PstI) 0.2-mL PCR tubes
Protocol	Into a 0.2-mL PCR tube add the following: 1 μg DNA 1 μL restriction enzyme 1 1 μL restriction enzyme 2 2 μL 10X appropriate buffer 1 μL 100X BSA, if using ddH₂O to a final volume of 20 μL Incubate at 37°C for one hour. Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes.

Ligation of DNA Inserts to Plasmid Backbones
Experimental Details and Rationale	Digested registry DNA or digested genetic parts from IDT were ligated to either pSB1c3 or pET29B for propagation in E.coli DH5ɑ or protein expression in E.coli BL21. Later, our parts were ligated to pSB1c3 for submission to the iGEM registry.
Materials	Digested vector DNA Digested insert DNA 10X DNA ligase buffer (from New England BIolabs) T4 DNA ligase (1 U/ μL) (from New England Biolabs) ddH2O 0.2-mL PCR tubes
Protocol	To a 0.2-mL PCR tube add: 50 ng digested vector DNA Appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector 1 μL T4 DNA ligase 2 μL 10X T4 DNA ligase buffer ddH2O to a total volume of 20 μL Incubate tube at room temperature overnight. Use 10 μL to transform cells, store at -20°C.

Preparation of Agar with Antibiotics
Experimental Details and Rationale	Antibiotics were added to agar to select for successful E.coli transformants. The vector pSB1c3 was selected for with chloramphenicol and pET29B was selected for with kanamycin.
Materials	Luria-Bertani broth with agar: 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract 15% (w/v) agar Appropriate antibiotic: kanamycin (final concentration of 100 μg/mL) chloramphenicol (final concentration of 30 μg/mL) dH2O 1500-mL Erlenmeyer flask Stir bar Aluminum foil
Protocol	In a 1500-mL Erlenmeyer flask add 10g tryptone, 5 g yeast extract, 10g NaCl and 15g agar. Dissolve solids in 1000 mL dH2O and add a stir bar. Cover flask loosely with aluminum foil, secure with autoclave tape and sterilize by autoclaving for 20 minutes. Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic. Stir on hot plate and magnetic stirrer for 30 seconds. Pour agar into plates using aseptic technique.

Plating Culture Broth on Agar Plates
Experimental Details and Rationale	Culture broth was plated on agar to isolate single colonies of E.coli.
Materials	Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod Bunsen burner
Protocol	Using aseptic technique pipette 50-100 μL of bacterial culture onto agar plate. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off. Incubate plates at 37°C overnight or until growth is observed.

Streaking Agar Plates
Experimental Details and Rationale	Culture broth was streaked on agar to isolate single colonies of E.coli.
Materials	Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod Bunsen burner
Protocol	Using aseptic technique pipette 50-100 μL of bacterial culture onto agar plate. Dip spreading rod in 70% ethanol, pass over flame, and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time. Dip spreading rod in 70% ethanol, pass over flame, and allow excess liquid to burn off. Incubate plates at 37°C overnight or until growth is observed.

References

Rose, C., Parker, A., Jefferson, B., & Cartmell, E. (2015). The Characterization of Feces and Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical Reviews In Environmental Science And Technology, 45(17), 1827-1879. http://dx.doi.org/10.1080/10643389.2014.1000761