Team:Brown/Notebook/Timeline

• Team Toxipop created a timeline back in May to follow throughout the summer.

Timeline as presented to Faculty Mentors and Graduate Advisors in May:

• Week 1: Obtaining DNA from Texas lab or outsourcing production. Run experiments testing known methods of lysis & resistance measurements—change procedure and apparatus as necessary.

• Week 2: Insert S-R-Rz cassette into plasmid and transform into bacteria. Screen bacteria to verify transformation success.

• Week 3: Create testing apparatus for resistance measurement. Begin modeling.

• Weeks 4-7: Experimentation

• Week 8: Analysis of Data, Graphing, Presentation

• Weeks 9 - 10: Allowance for any delays, further experimentation, begin second project.

Actual Timeline

Week One

• We were able to get the multiple strains from Vivek Jain and John Mekalanos at the Harvard Medical School.
• 1 pRG1 DH5alpha cells containing the S,R,Rz lysis cassette under a Plac promoter. Ampicillin Resistance.
• 2 pDKL02 S17-1 cells with the lysis cassette under an IPTG promoter (also containing the mob element). Kanamycin Resistant.
• 3 pVJ4 SM10 cells. Lysis cassette from RY100 cloned into pBAD18.mob 1. Amp Resistant.
• 4 pVJ13 DH5alpha cells clonded into pBAD33. Chloramphenicol Resistant.
• We worked with the Freeze-Thaw Method of Lysis in the few first days of summer. In addition, the FastLyse reagent was ordered but could not be used due to the significant changes in resistance caused due to the FastLyse alone. A change in resistance due to the intracellular ionic content of the E. coli bacteria could not be detected with the addition of FastLyse.

Week Two

• The S,R,Rz genes were provided already transformed. We spent most of week two working on two separate projects: 1. Testing lysis with Optical Density measurements and 2. Construction of a precise and accurate measurement apparatus.
• Multiple versions of the testing apparatus were made throughout the summer. Initial designs included legos, skewers, pipette tips, and copper electrodes. As the summer progressed, our apparatus became more complex. We quickly made the transition from standard voltmeter to a Data Acquisition Card coupled with LabView Computer Software. We quickly ditched the multi-stranded copper wires for Gold plated and Gold electrodes. In addition, we moved to an alternating current to prevent the ions from settling on the electrodes and affecting current.

Week Three

• Continued apparatus work in cooperation with Professor Jerry Daniels in Engineering and Daniel Ludwig, Biomedical Engineering '09.
• We made serial dilutions of salt water to standardize the apparatus. With this information we could determine the concentration of ions needed to cause a resistance change.
• We continued to perform Optical Density tests to determine how long it should take for lysis to occur.

Week Four

• PCR Primers ordered for PVJ4 in order to commence Biobricking procedures. Primers, however, did not work. Second and third sets were ordered.
• Continued apparatus work. Professor Tayhas Palmore gave us access to her lab to made PDMS (Polydimethylsiloxane) molds. The PDMS mold allowed us to secure the placement of our electrodes. Electrodes will remain in a fixed position approximately 1 cm apart.