McGill/August

From 2007.igem.org

Revision as of 18:58, 22 August 2007

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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

O.D antibiotics experiments results

A10 c-no antiobiotics : 0.047 B10 c-amp (1 ul/ml): 0.048 C10 c-amp (2 ul/ml): 0.052 D10 c-amp ( 5 ul/ml): 0.049 E10 c-amp (10 ul/ml):0.048 F10 c-amp (5 ul/ml): 0.048 G10 c-amp (10 ul/ml):0.046 H10 c-amp ( 20 ul/ml): 0.047

A11 c-kan (50 ul/ml):0.050 B11 c-kan (100 ul/ml):0.048 C11 J-amp (1 ul/ml): 1.264 D11 J-amp (2 ul/ml): 1.142 E11 J-amp (5 ul/ml): 1.143 F11 J-amp ( 10 ul/ml) : 1.162 G11 J-amp (1 ul/ml): 0.420 H11 J-amp( 2 ul/ml) : 0.350

A12 I-amp (5 ul/ml): 0.092 B12 I-amp ( 10 ul/ml): 0.494 C12 J-kan ( 5 ul/ml): 0.754 D12 J-kan (10 ul/ml): 0.152 E12 J-kan ( 20 ul/ml): 0.082. F12 J-kan (50 ul/ml): 0.184 G12 I-kan ( 5 ul/ml): 0.028 H12 I-kan(10 ul/ml):0.965

August 6

• Dilutions for a plate reader experiment:

1. 4 x I+J
2. 4 x I+J + AHL
3. 4 x I+J + Dox
4. 4 x I+J + IPTG
5. 4 x I+J + IPTG + AHL
6. 4 x I+J + IPTG + Dox

The above sample were then washed twice with Minimal Media and then suspended in supplemented minimal media with the correct concentrations of the about additives. Both concentrated (0.5mL) and dilute (1.5mL) were tested in the plate reader. Unfortunately, due to technical difficulties, there were no results to report.

• Seedings were also done for I+J to repeat the experiments above tomorrow. In addition, seeding of I5611 and I5612 was done to screen for the prsence of the bricks.

August 7

• Dilutions were conducted for the seedings from last night and grown to an OD of 0.3. Again, dilutions containing, AHL, Dox and IPTG were made to run in parallel with I+J. The samples were run in the Spectrophotometer for 18 hours to assay the division of cells through the optical density. Results indicate that the majority of cells had an exponential decrease in O.D. meaning that the cells are dying or simply clumping and settling to the bottom of the testing apparatus despite the shaking of the system.

• Miniprep of I5611 and I5612 by pooling three aliquots into one volume of buffer P1. Despite the super concentrating, the concentration of the DNA was still very low.

• The restriction digest from August 2 was repeated and the results obtained were similar. The I5612 showed no DNA to be present (no bands) whereas the bands for the I5611 indicated that the construct was indeed present.

August 8=

• Performed a large scale digest of I5611, pSB1AK3, which were found present, after we did a gel extraction of pSB1AK3, following protocol.