PROJECT

RESULTS

Day 44 - Monday, August 15th 2011

Miniprepped overnight cultures of the genomic promoter ligations.
PCRed the parts that were successfully sequenced because we didn’t have much DNA
Attempted to re-PCR the strep tag on. None of our transformations grew, even the ones where we used kinase, so we think the original PCR never worked.
Worked on hammering out the details of our human practices project
Developed a presentation of our math model
Began ligating our sequence confirmed PCR products onto the correct backbones.
Digested the RBS34+Parts minipreps from friday and ran them on a gel to confirm their validity.
Started a competent cell test

Day 45 - Tuesday, August 17th 2011

Prepared for a PAGE gel tomorrow to asses LasR and RhlR activity
Planned for testing at the HTA lab
Transformed yesterday’s backbone correction ligations.
Digested the Genomic Promoter + RBS composites, and ligated them with reporter genes.
Worked on catching up the few remaining parts that were not successfully sequenced, such as CP+RBS+RFP and CP+RBS+LasR.

Day 46 - Wednesday, August 17th 2011

All the backbone transformations succeeded! There were both red (original backbone) and white (correct product) on all the colonies.
Presented our math model to the advisers
Met with Sara at the high throughput lab to plan our testing, decided to run a control sample tomorrow.
Started growing up CP->RBS->Receptor cells for a PAGE gel and then sonicated them.
Transformed all of the genomic promoter ligations
Started overnight cultures for testing
Started overnight cultures (70!) of the backbone correction ligations.
Made Amp/Kan plates (among others) so that we could try a double transformation with the seperate construct plasmids if we want to or if putting them on the same plasmid caused any problems.

Day 47 - Thursday, August 18th 2011

Completed 70 minipreps of our backbone correction ligations!
Ran a PAGE gel to asses LasR and RhlR activity
Made new SOB and plates
Our cells were not growing properly in the m9 media, so we made it again from a different recipe. This has forced us to delay our testing.
Analyzed a recently published paper (yesterday!) that provides a lot of relevant information about our project.
Ligated our strep tag parts
Stored the genomic promoter transformations in the cold room. They looked good, but we are postponing overnights to catch up on everything else.

Day 48 - Friday, August 19th 2011

We digested all 70 minipreps from yesterday with the X and P enzymes. In the afternoon, we ran all 70 digests on gels to confirm that our constructs have been correctly inserted into their final backbones.
We got the results of our PAGE gel
We tested samples to calibrate the differences between our spectrophotometer and the High Throughput Lab plate reader.
We finished remaking M9 media.
Transformed Strep Tag Ligations