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.

8/7/10 notes

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.

8/9/10 notes

8/11/2010

Kevin, Marc

Met with Chris, received advice for updating digest and ligation protocols.

8/12/2010

Kevin, Marc, Alena Met and discussed protocols for digestion and ligation od FIMB into pBAD.

1. Added 5 mL of LB broth each to 2 50 mL falcon tubes in the ERB lab using sterile technique. 2. Added 5 microliters of Kanamycin to each of the 50 mL tubes in step 1.

Went to the budget committee meeting for 1 hour with the tubes.

3. Obtained the cryostock of pBAD from the Lin -80C freezer (iGEM box cell #73) 4. Stabbed cryostock using a sterile 200 microliter pipette tip and pipetted into media from step 2. 5. At 8:05PM placed the two falcon tubes from step 4 into the incubator/shaker at 30C.

In the Lab