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.

Week Five

• Continued work with the apparatus. An amplifier was added.
• Multiple tests with our PVJ4 plasmid were conducted, each one having a different concentration of engineered bacteria. Solutions were concentrated up to 6X.
• We also experimented with LB Lennox, Luria, M9 Minimal Media, Distilled/Sterile Water, as well as Milli-Q water. Cells were pelleted and resuspended in one of the aforementioned liquids. Cells grew the best in LB Lennox due to the greater concentration of ions and bacteria cell nutrients. (LB Lennox - 5g NaCl/Liter LB Luria - .5g NaCl/Liter) We had to take into consideration osmotic gradients especially with non-ionic Milli-Q Water.

Week Six

• Apparatus work with Daniel Ludwig and Professors Tayhas Palmore and Jerry Daniels.
• PCR work with the PVJ4 plasmid in order to Biobrick.

Week Seven

• Began to explore other ideas for a reporting system. Biological means of detection included placing a Green Fluorescent Protein under the control of the same inducible promoter that controls the Cell Lysis Cassette. We also considered an AND Gate where the S and R genes have different promoters. This would allow us to test for multiple toxins within water samples.
• Previous PCR's have been unsuccessful. Ordered "Middle" primers that started within the sequence of the pVJ4 Cassette. The initial and final 100 base pairs of the Cassette would be within the amplified sequences.

Week Eight

• UTRA Symposium at Brown. Team Toxipop shared the summer's research with the Brown scientific community.
• Continued sensitivity testing of our the apparatus with Daniel Ludwig.
• Different concentrations of bacterial cells tested with varying amounts of Arabinose. The control of these tests was either a pVJ4 culture without added Arabinose or a plasmid containing the Cell Lysis Cassette under a different promoter, for example pRG1 - on a Plac plasmid.

Week Nine +

• Wrap up of the summer. Work resumed in September.
• More sensitivity testing with NaCl and Milli-Q water.
• Made the switch to conductivity measurements. Obtained a conductivity probe and obtained beautiful results. Cells were not concentrated. Arabinose added and probe left in solution for four hours. A steep curve resulted!
• Much of our efforts were concentrated on Biobricking our different parts. Team Toxipop had problems over the summer obtaining PCR products. We were finally able to Biobrick the pVJ4 plasmid, the S105 gene, and the lysis cassette with S105,R,Rz genes. The pVJ4 plasmid contains the Cell Lysis Cassette under the control of an Arabinose Inducible Promoter. All tests run throughout the summer were done with pVJ4. The team received the S105 cassette and gene in September from Ry Young's Lab at Texas A&M.