PROJECT

RESULTS

Day 6 - Monday, June 20th 2011

Prepared all relevant buffers for competency procedure
Started growing our seed stock over night so that we could perform the competency procedure in the morning.
Obtained the 2011 distribution kit complete with DNA, plasmid backbones, pins and stickers!
Started a new transformation procedure with Part BBa_R0061 from the 2011 kit. We are mostly testing our transformation technique to make sure we are doing everything correctly.
Made a lot of progress finalizing the design of our project. We figured out what parts our project will need and determined where we will obtain each part.

Day 7 - Tuesday, June 21st 2011

The transformations from yesterday appeared successful.
The TOP10 cells we started growing last night were already at an OD600 level above 0.3, so we immediately proceeded to the next steps in the competency procedure. After a couple of centrifuging and resuspending cycles, we stored the competent cells in the -80 freezer.
We held a meeting with our advisers and presented our idea for a quorum-sensing based Psuedomonas aeruginosa detector. The advisers seemed to like the idea, and we are moving forward with it. We may also integrate sensing of other bacteria such as salmonella and staph depending on how the project goes.
Investigated the process of planning our parts. We explored the sandbox tool on the registry and began to use tools like restriction enzyme mappers to make sure the parts would work properly.

Day 8 - Wednesday, June 22nd 2011

Spent the morning making plates. We made 20 plates with each of the four main antibiotics.
Started another round of transformation procedures, this time with actual parts that we will use in our project.
Mapped the restriction sites and enzymes that we will need to use in order to get the final project up and running.

Day 9 - Thursday, June 23rd 2011

The transformations from last night appeared successful. There were many transformed colonies, although still slightly less than you would expect from fully competent colonies for our positive controls.
The promoter/RFP plasmid colonies were clearly red by lunchtime.
Finished analysis of the restriction sites, codon usage, and mRNA folding for the genes from P. Aeruginosa we may use.
Finished cataloging and inventory of parts that may be useful to our project.
Began transformations of parts we may use that came in the 2011 distribution kit.
Started overnight cultures with our transformed bacteria.
Began to store DNA from the used wells in the distribution kit in microcentrifuge tubes to ensure preservation.
Started another batch of competent cells

Day 10 - Friday, June 24th 2011

The transformations appeared successful. All of the plates except one had colonies.
The competent cells once again grew faster than we were expect and had OD600 levels above 0.3 by the morning. We diluted the cells in order to lower their OD closer to the target level before proceeding with the rest of the procedure. We’ve also learned to aliquot smaller volumes of the cells because we only use 25 microliters per transformation.
We began making a glycerol stock so that we can store our transformed bacteria.
We miniprepped the parts that we overnighted last night. When we were done, we tested the DNA concentration. The positive controls were both around 200-300 ng/uL and the part was about 65 ng/uL.
We started overnight cultures from the 5 successful transformations from yesterday.
On Saturday, we came in to transfer the overnight cultures into long-term glycerol stocks.