IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/07/20

July 20th, 2009

Checked Rob's plate. A few single colonies, little bit of mold (not too bad) Should replate anyways

- Kan-GFP-Paraoxon BS looks good. Colonies starting to grow.

                   OD of O/N delta35 cultures (5) = 1.6

-dilute to OD 0.5 (~ 30 fold dilution) @ 10:00

                   100 ul / 3 ml culture

- Should be ready (OD ~ 0.2) at 1 p.m

- Checked OD's are 0.2-0.26 except for culture 3. will leave in incubator and initiate biosensor

Culture: 1

                  [] Paraoxon = 1mM
                  ul of 20 mM Paraoxon = 50ul
                  uL cell culture = 950 ul
                  total = 1 mL

Culture: 2

                  [] Paraoxon = 1mM
                  ul of 20 mM Paraoxon = 50ul
                  uL cell culture = 950 ul
                  total = 1 mL

Culture: 4 - analogous formulations to Culture 1 and 2 Culture: 5 - Same formulation as 1,2,4

Culture 1(II)

                  [] Paraoxon = 2mM
                  ul of 20 mM Paraoxon = 100ul
                  uL cell culture = 900 ul
                  total = 1 mL

Culture 1(III)

                  [] Paraoxon = 3mM
                  ul of 20 mM Paraoxon = 150ul
                  uL cell culture = 850 ul
                  total = 1 mL

Check in 1 hour (put in at ~ 1:45, 30 degrees C, rotating). - 2:45

Rob's OD for starter cultures (20 ml total) @ 1 PM: 0.15

- At about 4 check for GFP. Couldn't see anything. Used fluorescent microscope in kron lab

- faint glow in culture 5. Will let all cultures grow another 1 hour

Note: 2mM and 3mM cells seemed to have a lot more dead cells. Could paraoxon be inhibiting growth? The schofield paper says it shouldn't (at least for normal cultures).

Culture 5: seemed to have faint glowing colonies. Viewed using fluorescent microscope(s) in Kron lab.

-40x, oil immersion.

- Pictures saved on wiki

Negative control: dead cells will glow as well, but will do so under all filters (UV, FITC)

- Should see glow only under GFP filter

- We will restreak the restreaks tomorrow on YPD.

- Also use same colony for O/N culture. For...

                  - glycerol stock
                  - GFP test
                  - confrmation of integration using gene and Kan-casette specific primers

-Does paraoxon inhibit growth? Test tomorrow. Using leftover culture 5 as starter

End of the Day