Experiments

Check out the protocols we used throughout our project

Cloning

We used these protocols to create plasmids with our genes of interest from genomic DNA, and transform those plasmids into E. coli

Centrifuge incoulated cell culture for 15 minutes at 5000g
Discard supernatant and resuspend in 1 mL of cloning water
Transfer solution into bashing bead lysis/filtration tube
Add 6 mL of fungal/bacterial DNA binding buffer
Vortex for 5 minutes
Spin down for 5 minutes at 5000g
Transfer filter to a 50 mL tube
Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
Add 200 uL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
Transfer column to a 1.5 mL microcentrifuge tube and add 30 uL of cloning water
Let sit for 4 minutes, then spin for 4 minutes at 10,000g
Measure concentration of gDNA and label tube

Add the following to a single PCR tube:

20-21.5 uL Cloning Water (depending on type of amplicon)
10 uL of Betaine
10 uL of 5x High-Fidelity Buffer
4 uL DMSO
2.5 uL 10 uM combined forward/reverse primers
1 uL DNTPs
0.5 uL of plasmid or 2 uL of gDNA
0.5 uL of Phusion Polymerase (kept on ice)

Immediately run in a thermocycler for the following times/temperatures

Initial Denaturation 98°C 30 sec
25-35 cycles

98°C 5-10 sec
45-72°C (at annealing temp) 10-30 sec
72°C 15-30 sec/(length of amplicon in kb)

Final Extension 72°C 5-10 minutes
Hold at 4°C

Store at 4°C until use

Gel Electrophoresis with SYBR safe (Moon Lab)

Making the Gel

Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA)

We generally made gels with 75 - 125 mL of TAE

Microwave in a covered erlenmeyer flask for 120 seconds
Add 1 uL of SYBR Safe or Ethidium Bromide per 20 mL of TAE
Pour into gel casing
Remove bubbles with a pipette tip
Insert well comb
Allow to set (~20 minutes)

Running the Gel

Remove well comb
Place gel in electrophoresis apparatus with the wells by the cathode (black)
Fill apparatus with TAE buffer so that the gel is completely submerged
Load wells with 10-60 uL of 5 (PCR product): 1 (6x Loading Dye)
Load 10 uL of ladder into first and last wells
Run at 125-130 V for 25-30 minutes

Adapted from Zymo Research Zymoclean™ Gel DNA Recovery Kit

Cut band from gel while minimizing time under UV light
Place gel in a microcentrifuge tube with 3x volume of Agarose Dissolving Buffer
Heat at 55°C until gel dissolves (~20 minutes)
Add to gel purification filter placed inside a collection tube
Centrifuge for 1 minute at 16,000 g and discard flow-through
Add 200 uL of DNA wash buffer, centrifuge for 1 minute at 16,000 g, discard flow-through, and repeat once
Centrifuge for 2 minutes at 16,000 g and discard flow-through
Place filter in 1.5 mL microcentrifuge and add 20-30 uL of cloning water
Wait for 4 minutes, then spin for 4 minutes at 16,000 g
Measure concentrations and label

Adapted from Zymo Research DNA Clean & Concentrator™-5 Kit

Add 50 uL DNA and 250 uL DNA Binding Buffer to a spin column in a collection tube
Centrifuge for 30 seconds at Xg and discard flow-through
Add 200 uL of DNA wash buffer, centrifuge for 30 seconds at 16,000g, discard flow-through,
and repeat once
Place filter in 1.5 mL microcentrifuge and add 20-30 uL of cloning water
Wait for 4 minutes, then spin for 4 minutes at 16,000g
Measure concentrations and label

In a PCR tube, combine:

0.4uL Cutsmart Buffer
0.4uL DpnI
plasmid backbone (either post PCR or gel extract)

Incubate at 37°C for 1 hour
DNA purification for post-DpnI mix to retrieve plasmid backbone
In a PCR tube, combine:

100 ng of plasmid backbone
Equimolar amount of each assembly piece
1.5 uL of restriction enzyme buffer
1 uL of ligase buffer
1 uL of Type IIs restriction enzyme
1 uL of T4 ligase
Cloning water to equal a total of 15 uL

Run in a thermocycler for the following times/temperatures:

50 cycles

3 minutes at 37°C
4 minutes at 16°C

1 cycle

5 minutes at 50°C
20 minutes at 80°C

Hold at 4°C

Transform into competent cells and/or store at -20°C

Electroporation

Defrost competent cells on ice for 5 minutes
Pipet 1.5 uL plasmid to the tube of competent cells
Pipet the mixture into an electroporation cuvette

Avoid touching the metal plate of the cuvette and introducing bubbles

Shock (Settings?)
Quickly add 500 uL of Lysogeny Broth (LB)
Incubate in a culture tube for 1 hour at 37°C
Pipet desired amount of solution onto a plate and spread evenly
Allow solution to dry and incubate for 16-18 hours
Wrap plate in parafilm and store at 4°C

Chemical Transformation

Thaw 100 uL of competent cells on ice
Add 100 ng of plasmid to cells
Incubate on ice for 20-30 minutes
Heat shock the cells for 60 seconds at 42°C
Return the cells to ice for 2 minutes
Add 300 uL of LB or SOB medium
Incubate in a culture tube for 1 hour at 37°C
Pipet desired amount of solution onto a plate and spread evenly
Allow solution to dry and incubate for 16-18 hours
Wrap plate in parafilm and store at 4°C

Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit

Spin down cells for 15 minutes at 3200g
Discard flow-through and resuspend in 600 uL of cloning water
Transfer to a 1.5 mL microcentrifuge tube
Add 100 uL of 6x Lysis Buffer and invert
Add 300 uL of cold Neutralization Buffer and mix thoroughly
Microcentrifuge for 4 minutes at 16,000g
Pour supernatant into a miniprep filter placed in a collection tube
Microcentrifuge for 1 minute at 16,000g and discard flow-through
Add 200 uL of Endo-wash buffer
Microcentrifuge for 30 seconds at 16,000g and discard flow-through
Add 400 uL of Zyppy Wash buffer
Microcentrifuge for 30 seconds at 16,000g and discard flow-through
Microcentrifuge for 2 minutes at 16,000g and discard flow-through
Place filter in 1.5 mL microcentrifuge and add 20-30 uL of cloning water
Wait for 4 minutes, then spin for 4 minutes at 16,000g
Measure concentrations and label

Add 4 mL of Lysogeny broth (LB) and 4 uL of each needed antibiotic to a culture tube
Add cells to the culture tube

If using frozen stock, scrape a small amount of stock with a pipette tip
If using a plate, carefully scrape a single colony with a pipette tip

Grow at 37°C for 16-18 hours

BioBrick

We used these protocols to manipulate BioBricks provided in the iGEM distribution kit and to create our own BioBricks from experimentally tested genes

Add the following to a PCR tube

29uL of miniprepped plasmid
11uL Cloning water
5uL of NEB Buffer (depends on REs used)
5uL total of Restriction Enzymes (SpeI, PstI, EcoRI, or XbaI)

Set at 37°C for 3 hours
Heat inactivate at 80C for 20 minute

Add the following to a PCR tube

100ng of backbone
3:1 ratio of insert:backbone
2uL 10X T4 DNA Ligase Buffer
1uL T4 DNA Ligase
Add Cloning water to get total volume of 20uL

Set at room temperature (~23°C) for 1-3 hours
Heat inactivate at 80°C for 20 minutes

Measurement and Assays

We used these protocols to test the effects of our plasmid constructs and quantify those results

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Adapted from Thermo Fisher Scientific Molecular Probes® ATP Determination Kit

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step 3

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Adapted from Vector Laboratories Quant*Tag Biotin Quantitation Kit

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Miscellaneous

These protocols served a variety of important purposes

Streak cells from frozen stock onto LB plate and incubate overnight at 37°C
Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37°C
Dilute 1 mL of culture into 50 mL LBMg (with antibiotic if applicable) pre warmed to 37°C
Grow culture in 37°C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)
Incubate cells for 20 minutes on ice
Transfer the cells to ice-cold sterile 50 mL tube
Centrifuge for 10 minutes at 3000 rpm at 4°C and discard the supernatant
Gently resuspend the cells in 20 mL of ice-cold TFB1 (name) with chilled pipet tips
Incubate cells on ice for 25 minutes
Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice
Resuspend cells in 4 mL of ice-cold TFB2
Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen
Store at -80°C

Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar
Fill to 500mL mark with Deionized water
Add a magnetic stir bar and mix thoroughly using a stir plate
Autoclave
Let cool, add 500 uL of (each) antibiotic, and stir using stir plate
In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry
Store at 4°C