"
Team:Michigan/Pili Expression
From 2010.igem.org
| Sunday | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | |
| Week 1 | - | 6/28/2010 | 6/29/2010 | 6/30/2010 | 7/1/2010 | - | - |
| Week 2 | - | - | - | 7/7/2010 | - | - | - |
| Week 3 | - | - | - | - | - | - | - |
| Week 4 | - | - | - | 7/21/2010 | - | - | - |
| Week 5 | - | - | 7/27/2010 | - | - | - | - |
Pili Expression Team
This team includes Marc Singer, Kevin Joseph, and Alena Wu.
6/28/2010
Made a 500 mL batch of LB broth
- When pouring in distilled water, pour a few mL at a time to avoid clumping.
- It takes 20g of powder to make 1 L of broth
Sterilized broth using the autoclave
- The temperature setting on the autoclave is off by a little bit
- Set dial 2 notches below 134°C.
6/29/2010
Started growing E. coli K12 cultures
- Poured 2 mL of LB broth into a Falcon tube
- Used strain of K12 from Dr. Lin's freezer
- Placed in incubator shaker for 24 hrs.
Added and inventoried supplies from Dr. Pinto's lab.
- Regular trash can be thrown out by going down one floor, then going outside to the trash bins.
- Chemical wastes must be cleaned by calling OSEH.
6/30/2010
Cryopreserved stock of K12
- 1 mL located in the iGEM box in the Lin lab.
- 1 mL located in the lab freezer.
7/1/2010
Cryopreserved DH5α according to protocol procedure on 6/30/2010
- Put 1 stock in -20°C fridge in 1239
- Put the other stock in the -80°C fridge in the Lin lab
7/7/2010
Obtain genomic DNA of CFT073 E. coli strain from Dr. Mobley's Lab
- stored in -20°C fridge on ice
7/21/2010
Kevin, Marc, Alena
Met in Dude to determine sequence of fim operon. Arranged meeting with Dr. Mobley's group next Tuesday to learn more about hyperpiliation and the cloning process.
7/27/2010
Kevin, Marc, Alena
Met with Chris Alteri from Dr. Harry Mobley's research group to discuss the best route to hyperproduce the pili. Chris recommended that we create a plasmid by cloning FimB into pBAD, and then inserting that plasmid in MG1655. In theory, that should activate flocculation in the E. coli, inducible by arabinose. Chris was able to give us the procedures for creating a plasmid with FimB, as well as the procedures for knocking out a gene.
In order to test how effectively the pili flocculate, we are planning to create an E. coli strain with fimE knocked out.
8/7/2010
Kevin, Marc, Alena
PCR #1 Used a gradient from 40C to 60C for the first 3 cycles to find the optimum anneling temperature.
All of the annealing temperatures gave a good result according to the gel.
8/9/2010
Kevin, Marc, Alena
Used a 57C degree annealing temperature to get enough DNA for the digest and ligation.
4 out of the 5 PCR reactions worked well according to the gel.
