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.
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.
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.
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.
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 or single isolated colony on agar plate
Inoculation loop
Bunsen burner
Protocol
Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on ¼ of the surface of agar in a zigzag motion.
Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1/4 of the surface of the agar.
Repeat Steps 3 and 4 two more times.
Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.
Incubate plates at 37°C overnight or until growth is observed.
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.
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.
Experimental Details and Rationale
Fragments of DNA are separated by size on the gel. This was used to visualize the results of restriction digests, particularly those done to confirm ligation or transformation.
Materials
TAE buffer:
40 mM Tris, pH 7.6
20 mM CH₃COOH
1 mM EDTA
Agarose
250 mL-Erlenmeyer flask
RedSafe Nucleic Acid Staining Solution
Gel casting tray and comb
10X loading dye
DNA sample
Microwave
Protocol
For a 1% gel (standard), add 0.3 g agarose to 30 mL TAE buffer in a 250 mL-Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).
Allow flask to cool in fumehood until warm to the touch before adding 1.5 μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.
Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.
Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.
Load samples of 11 μL DNA containing 1 μL loading dye.
Run gel at 100 V for 30 minutes or until loading dye is 1/2 way down the gel.
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.
Experimental Details and Rationale
Chemically competent DH5 Alpha and BL21 E. coli cells were prepared, which enabled them to be transformed with recombinant DNA.
Materials
Luria-Bertani broth:
10% (w/v) tryptone
5% (w/v) NaCl
10% (w/v) yeast extract
MgSO4
KCl
250 mL-Erlenmeyer flask
16x125 mm culture tubes
Spectrophotometer
Centrifuge
50-mL Falcon tubes
100 mM CaCl₂
glycerol
1.5-mL microcentrifuge tubes
Protocol
Subculture strain
1:50 subculture into 50ml LB(with 10mM MgSO4 and 1mM KCl)
28C shake to OD600 = 0.3 - 0.4
Chill on ice at least 10min
Put into 50ml pre-chilled tube, centrifuge 2500g x 8min, at 4C
Re-suspend in 10ml ice-cold 100mM CaCl2, gently mix on ice, incubate on ice for 10min to ON
Centrifuge 2500g x 8min, at 4C
Re-suspend in 500ul 100mM CaCl2 +10% glycerol, gentle pipetting up and down a few times, on ice at least 10min
Separate to 1.5ml pre-chilled tubes, 50ul cells each tube (use the large tips to separate it, always on ice)
Experimental Details and Rationale
Chemically competent E.coli DH5α was transformed with pSB1c3 or pET29b containing our genetic parts in order for that plasmid and part to be propagated. Chemically competent E.coli Bl21 was transformed with pSB1c3 or pET29B containing our genetic parts in order for those proteins to be expressed.
Materials
Competent E.coli aliquots (200 μL )
DNA for transformation
Luria-Bertani broth or SOC Media
Agar plate with appropriate antibiotic
Protocol
Thaw 200 μL aliquot of competent E.coli DH5α cells on ice just before use.
Add 0.3-1 μg DNA to cells (in maximum 20 μL), flick gently to mix, and place on ice for 30 minutes.
Heat shock for 60-75 seconds at 42°C.
Place on ice for 5 minutes
Add 250 μL Luria-Bertani or SOC medium to aliquot of cells.
Incubate cells for 60 minutes at 37°C, shaking at 200 rpm for 1 hourv
Pellet cells in a microcentrifuge at 3500 g for 1 minute and discard supernatant.
Resuspend pellet in 250 μL Luria-Bertani broth.
Plate 50-100 μL of resuspended culture on agar plate with appropriate antibiotic and spread.
Incubate plates at 37°C overnight or until desired growth is observed.
Experimental Details and Rationale
Glycerol stocks of transformed E.coli were prepared for long-term storage of the cells at -80°C.
Materials
Overnight culture of transformed bacteria
Sterile 1.5-mL cryo-tubes
Sterile 50% glycerol
Protocol
Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5-mL cryo-tube.
Using aseptic technique add 0.5 mL of overnight culture.
Pipette up and down gently to mix.
Store at -80°C for up to 1 year.
Experimental Details and Rationale
Phosphorylated ends of DNA and RNA were removed, preventing unwanted ligation of linearized DNA.
Materials
Digested DNA vector
Antarctic phosphatase buffer
Antarctic phosphatase
ddH2O
0.2-mL PCR tubes
Protocol
To vector tube from restriction digest, add:
1 μL 10X Antarctic phosphatase buffer
1 μL Antarctic phosphatase
8 μL ddH₂O
Incubate tube at 37°C for 30 minutes.
Deactivate Antarctic phosphatase via heat shock by incubating tube at 80°C for 20 minutes.
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
