Brown: Team Resistance/8 July 2008

==

Cultures from last night taken out 9am Made 1/100 dilutions of the pVJ4 and pRG1 plasmids and incubated from 9:15am to 11:15am (until they reached mod-log phase)

Went to Palmore lab to make a PDMS mold in which we would secure our wires. This mold would prevent the wires from moving thus ensuring the distance between the wires remains constant and that our resistance readings are more accurate

• Made a mold of a 6-well plate lid
• To create holes in the PDMS so that the wires can later be slipped through, we taped capillary tubes upright in the lid (2 pieces of capillary tubes taped upright approximately 1 cm apart in above each well in the plate, so that two wires can be slipped through the mold and into each well for experimentation)
• The PDMS was poured into the lid and made its way around the capillary tubes

To create mold

• mix setting agent and elastiomer mix (PDMS)
• centrifuge @1000rps for 5 minutes
• pour into mold
• cure for 1 hour at 70degress C (oven in Palmore lab)
• Pull capillary tubes out and insert wires surrounded by Teflon tubing (provided by Ed Mullen, they are the same diameter as the capillary tubes)

Dan came back to the iGEM lab with us to help us create the Labview program Rough diagram of circuit drawn by Dan on pg __ of Rima’s iGEM notebook

When we returned to lab, we added arabinose to the pVJ4 dilutions created earlier to gauge the exact time needed for lysis pVJ4 dilution 1: control pVJ4 dilution 2: added 0.0110g arabinose to the 5mL culture (0.2% by weight)

-->OD measurements: Time pVJ4-1 (control) pVJ4-2 3:00pm 0.084 0.063 4:10 0.082 0.077 5:10 0.083 0.081

No lysis has occurred yet so added 0.019g arabinose, adding arabinose to 0.4% of the solution

-->7:00 0.080 0.045

Did see lysis after approximately 4hrs. Is it because of the extra arabinose or the length of time?

Observed that when cells lyse, the solution clears and its appearance is similar to LB→ the R protein degrades the cell wall

7/11/2008

Ran restriction digest of both pDKL02 (IPTG-induced promoter) and pVJ4 at the EcoRI and EcoRV sites (should cut halfway through the cassette)

• Mini-prepped the pDKL02 DNA
• Only saw band for pDKL02 but assumed that it must also be there in the pVJ4 but something went wrong

This week we did a lot of work with the apparatus because it was knocked over and disconnected and when reconnected, it did not give consistent readings at all