From 2007.igem.org

August 2007
We	Th	F	Sa	Su	M	Tu
1	2	3	4	5	6	17
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31

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August 2007

August 1

• Dilutions of all the seeding yesterday intended for imaging todya on the plate reaser where done. For the oscillator (I+J), only BL21 cells grew thus the experiment shall be carried out thusly and compared to previous results in the MC4100 cells. 350uL of culture was diluted to 5mL of minimal media making a total of 16 dilutions. Of these, to 4 of them, AHL (1uL/mL) was added and to a further 4, DOX was added (1uL/ml). At an OD of 3, each set of two dilutions were washed into one epitube and made into both a concentrated (0.5mL final volume) and a dilute (1.5uL final volume) and run on the plate reader for 18 hours.

• Results from the antibiotic experiment were nonsense thus a repeat must be made. The following ODs of the sample are listed below:

Control, no antibiotics: 1.643

• Control Kan (no colony)
• 5 uL/mL 0.744
• 10uL/mL 1.080
• 20uL/mL 1.109
• 50uL/mL 1.126
• 100uL/mL 0.841

• I in Top10 in Kan
• 5uL/mL 1.133
• 10uL/mL 1.188
• 50uL/mL 1.143
• 100uL/mL 0.744

• J in Top10 in Kan
• 5uL/mL 1.080
• 10uL/mL 1.109
• 20uL/mL 1.126
• 50uL/mL 0.841

• Control Amp (no colony)
• 1uL/mL 1.133
• 2uL/mL 1.188
• 5uL/mL 1.143
• 10uL/mL 1.121

• I in Top10 in Amp
• 1uL/mL 1.072
• 2uL/mL 1.254
• 5uL/mL 1.028
• 10uL/mL 1.092

• J in top10 in Amp
• 1uL/mL 0.194
• 2uL/mL 0.080
• 5uL/mL 1.030
• 10uL/mL 0.199

Obviously, there must be contamination in most of the samples as the controls with and without antibiotics have significant growth and the concentrations of the trial samples are relatively similar. A repeat of the experiment shall be implemented soon.

August 2

• restriction digest of I5611(LVA) and I5612 ( no YFP)

for I5611 (6.3 kb) Xua I and Pst1 were used in buffer 2+ BSA Pvt I was used for linear cut in buffer 3 +BSA Eco RI and BsrGI were used to in buffer 2 + BSA

for I5612 EcoRI and AfeI were used in buffer 4 + BSA Apa and PstI were used in buffer 4 +BSA

• Transformation of I+J and J in BL21
• antibiotics experiments

Kan control :5,10,20,50,100 ul/ml I in Kan : 5,10,50,100 ul/ml J in Kan : 5,10,20,50,100 ul/ml

Amp control : 1,2,5,10 ul/ml I in Amp : 1,2,5,10 ul/ml J in Amp : 1,2,5,10 ul/ml