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Public Engagement and Education

“Synthetic Biology” can sound daunting to even the bravest of academics. We want to change that. Removing the stigma around science might not be accomplished in one iGEM season, or even by one iGEM team, but we made a difference this year...and that’s something to be proud of. We taught high schoolers about bioengineering to help them gain confidence in their ability. We played with lots of kids to inspire them to pursue science. We threw a party to tell people all about iGEM and what biology can do. We made an app that will introduce people to plasmids wherever they are. Most of all, we told everyone we met how much is possible thanks to synthetic biology.

Bioengineering Camp

Materials:
LB broth
Ice
Selection plates

Methods:

Thaw 50µL competent E. coli cells on ice for 10 minutes
Add:
- 5-10 µl DNA from a ligation reaction mix or
- 10-100ng DNA of a known plasmid
Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 µl of room temperature SOC or LB media into the mixture.
Incubate at 37°C and 200-250 rpm for 60 minutes.
Mix the cells thoroughly by flicking the tube and inverting.
Spread:
- For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
  1. Pellet cells at 8000rpm for 3 minutes
  2. Remove and dispense 600 µL of supernatant
  3. Re-suspend cells by light vortexing
  4. Plate resuspended cells as above
- For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
  1. Pellet cells at 8000rpm for 3 minutes
  2. Remove and dispense 600 µL of supernatant
  3. Re-suspend cells by light vortexing
  4. Plate resuspended cells as above
Incubate overnight at 37°C with plates upside down.

Visit to Sherman Elementary

Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner

Add 5 ml liquid LB media into 12 ml culture tubes
Add 5 μl of appropriate antibiotic into the broth
Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm

Sue’s Tech Kitchen

Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA

Methods:
For a 25 µL reaction

In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
Gently mix the reaction
If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
Transfer the PCR tube from ice to a PCR machine to begin thermocycling

For a 50 µL reaction

In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
Gently mix the reaction
If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling

Thermocycling
The PCR machine should be set to run the following steps:

Step	Temperature (°C)	Time
Initial denaturation	98	30 seconds
25-35 cycles	98 (denaturation) 45-72 (annealing) see Note 1 72 (extension)	5-10 seconds 10-30 seconds 15-30 seconds per kb
Final extension	72	2-5 minutes
Hold	4	Indefinitely

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/

Launch Party

Materials:
Sterile Water
25 µL RedTaq mastermix
1 E. coli colony
2.5 µL of 10 µM forward primer
2.5 µL of 10 µM reverse primer

Methods:

Add a single colony of cells to 50 µL of water. Incubate at 95C for a minute to lyse the cells.
Combine 1 µL cell lysate, 25 µL RedTaq mastermix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and sterile water up to 50 µL.
Note: It is important to add RedTaq Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
Incubate in the thermocycler - Taq has a lower optimum temperature than Phusion.

Thermocycling
The PCR machine should be set to run the following steps:

Step	Temperature (°C)	Time
Initial denaturation	98	30 seconds
25-35 cycles	98 (denaturation) 45-72 (annealing) see Note 1 68 (extension)	5-10 seconds 10-30 seconds 15-30 seconds per kb
Final extension	72	5-10 minutes
Hold	4	Indefinitely

Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.

Our App

LB Broth
Materials:
25 g LB broth (powder)
1 Litre Purified Water

Methods:

Add 25g LB broth to 1 litre purified water
Autoclave

LB Agar
Materials:
37 g LB Agar (powder)
1 Litre Purified Water

Methods:

Add 37g LB Agar to 1 litre purified water
Autoclave

Glycerol Stocks
Materials:
500µl glycerol (80%)
500µl overnight culture in LB

Methods:

Add 500µl glycerol (80%) to 1.5ml eppendorf tube
Add 500µl overnight culture in LB
Store at -80°C

Social Media

Materials:
Agarose Powder
TAE buffer
Gel mould
5-10 µL SybrSafe
Gel Tank
8-10 µL DNA ladder
DNA loading dye

Methods:

Prepare 8% w/v solution of agarose powder in 1/10 TAE buffer (e.g. 0.8g agarose powder in 100 mL buffer) using a conical flask
Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
Pour the solution into a gel mould
Add 5-10 µL SybrSafe® to the solution. Make sure there are no bubbles in the solution.
Allows the solution to set (approx 15-20 minutes)
Transfer the agarose gel to a tank, remove the comb and apply:
- 8-10 µL of the DNA ladder
- DNA samples with the corresponding amount of DNA loading dye (6X)
Run the gel for 45-60 minutes at 100V

Pacific Northwest Meetup

Materials:
Microcentrifuge tube
Ice
1 µL T4 DNA Ligase
2 µL 10X T4 DNA ligase buffer
50ng Vector Plasmid
Insert DNA
Sterile water

Methods:

Calculate volumes of vector and insert DNA required using NEBioCalculator ( http://nebiocalculator.neb.com/#!/ligation - required molar ratio of 1:3::vector:insert)
Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last)
Make reaction up to 20 µL using sterile water
Incubate at room temperature for 30 - 60 min for sticky ends or 1-2 hours for blunt ends

Clover Park Rotary Club Presentation

Materials:
Restriction Enzyme: NEB enzyme finder used to see which restriction enzymes are required
10X buffer: NEB double digest finder used to see which buffers are required for the particular restriction enzymes
Plasmid DNA
Sterile water

Methods:

Component	Test digest Double digestion (20 µL, for construct analysis)	Assemble digest Double digestion (20-50 µL, for gene assembly)
Sterile water	to 20 µL	to 50 µL
10X buffer	2 µL	2-5 µL
Plasmid DNA	~200 ng for test digest, (DNA mass is variable dependent on insert size. Smallest digestion fragment mass should be > 50ng)	1,000 -2,000 ng
Restriction enzyme	0.5 µL + 0.5 µL	1 µL + 1 µL

Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.
Incubate digestion reaction at 37°C:
1. 30 min for Test digest
2. 2-3 hours or overnight for Assembly digest
Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

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Team:Tacoma RAINmakers/Public Engagement