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From 2010.igem.org

9/23/10

Today I have been setting up for the trial of our part T9002 inside agrobacteria to see if in the presence of AHL it floureces.

1) I took and set up new growing Cultures of T9002 and E – Coli electro comp cells placed 5 mL into 50 mL sterile tubes one with Agro w/T9002 and one with E – Coli to make electro comp cells.

2) then drew off “2” 1 mL inciments of T9002 Sol. and placed 4.5 mL of Amp w/T9002

3) now we are ready to perform trial taking Clean 1.5 mL Centrifuge tubes labeling 1 – 6 the first contains 200 mL Argo/T9002 & 800 mL LB/Broth second w/200 mL Argo/T9002 w/800 mL Argobacteria Supernate. Third w/200 mL Argo/T9002 w/800 mL E – Coli Supernate (AHL) we are looking for a glow from this one Fourth 200 mL LB Broth – 800 mL LB Broth Fifth 200 mL LB Broth – 800 mL Argobacteria Sixth 200 mL LB Broth – 800 mL E – Coli Supernate

Then they will all be tested inside a florimeter. Dylan is running a Spec reading on Agrobacteria/and Agro/w T9002 to see if the concentrations are close to the same. then do the trial he found that absorbance was 4 times higher so Dylan diluted the sample of T9002 ¼ adding LB – Lennox to this gave a spec reading of .303 and org. was .333 a difference of about 10 percent so we believe that will be ok we will work with this . See Dylans Lab notebook for more accurate details.

for now I will be making and washing electrocomp E – Coli cells to be frozen

1) spinning off supernate from cells in 50 mL Centifuge

2) then add 1 mL 10 percent glycerol and remove place in 1.5 mL centrifuge tube I will split cells into 6 centrifuge tubes adding 1 mL 10 percent glycerol and spin inside cold centrifuge @ 1000x for 10 min.

3) then repeat a total of 4 times

4) then leave about 40 mL of glycerol into the tubes and freeze @ -80 degrees C until needed.

I took the electro comp cells I’m about to wash and ran a spec read I took 20 mL of E – Coli cells and placed into 980 mL of 10 percent glycerol then vortexed to mix then pipetted on absorbance of .15 50 20 mL into 1000w 1000/.20 = 50 X .15 = 7.5 so our OD is 7.5 mL so what I did was split the cells into 6 seperate tubes to be washed following protocol from pg20 while Dylan runs the Trial on T9002 from pg25 For results see pg 30