From 2007.igem.org

August 2007
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Contents 1 August 2007 1.1 August 1 1.2 August 2 1.3 August 6 1.4 August 7 1.5 August 8 1.6 August 9 1.7 August 10 1.8 August 12 1.9 August 21 1.10 August 22 1.11 August 23

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 and pSB1AC3 whose presence was confirmed. A gel extraction of the fragments was implemented to isolate the fragments.

August 9

• A first ligation was then preformed which would place the I5611 represillator into pSB1AC3, a medium copy number plasmid with amp/Cm resistance. The following ligation protocol was used:
  

30uL total
3ul 10X ligation buffer
1.5uL T4 DNA ligase
2 molar units insert I5611 (21.2uL)
1 molar unit vector pSB1AC3 (3.81uL)
The molar ratios are slightly deviated as the lengths of the insert and vector are different (4225bp and 3047bp respectively). The ligation was placed in a cool block at 16 degrees overnight.

• Also, six seedings were done for the J40001 brick only into BL21 cells to assay the possibility of oscillations without the presence of the I15001 brick to confirm the suspicion of the contributing factors of the BL21's endogenous Lac to the oscillations seen last year.

August 10

• A tranformation of the ligation DNA into Top10 cells was preformed for amplification. Here, 10uL of DNA was mixed with the cells and heat shocked for 2 minutes. The system was then grown on Amp/Cm plates and no colonies were present meaning the ligation didn't work.
• Dilutions (500uL culture --> 5 mL minimal media) of the seeding were also preformed to the following manner:
  

4x J only
4x J + AHL
4x J + Dox
4x J + IPTG
4x J + IPTG + AHL
4x J + IPTG + Dox
After placing the above in a plate reader at two different concentrations (1.5mL and 3.0mL) oscillations were present in the J only, AHL only, and IPTG and AHL samples. The oscillations in the samples seem to generally decrease in flourescence and the dilute sample shows better results then the concentrated as before. Since these are more predicted results then the I + J when the samples were more concentrated, it is hypothesized that the more concentreated samples flooded the system with AHL and thereby, the cells could not respond to more AHL being added to the system and the addition of DOX which decreases the amount of AHL would actually enable communication to a higher degree. More experiments with the I + J at these concentrations and more dilute concentrations will be preformed shortly.

August 12

• First, new amp/Cm plates were made (1uL/mL amp and 0.8uL/mL Cm) to minimize the possibility that poor plates are responsible for the lack of growth of the pSB1AC3.
• A series of transformations was also conducted:

1. I15001 and J40001 into BL21

• 5uL each of DNA with a 2 minute heat shock

1. Ligation DNA into Top10

• 10uL of DNA with a 2 minute heat shock

1. pSB1AC3 into Top10

• 5uL of DNA with a 2 minute heat shock

• Results: Growth was observed on the ligation plates, the pSB1AC3 though not on the I + J plates. Since there was limited growth on the ligation plates, another ligation will be done though with better concentrations of DNA. It was found through optical density analysis that the concentration of DNA as very low and not even a nanogram of DNA could be put together for the ligation (thus suspecting a contaminent). So, we shall reseed the cells and preform a midi prep to further concentrate the samples and hopefully yield better ligation results.

August 21

• A series of seedings were preformed:

1. E0434 into LB
2. I5611 into LB
3. Ligation mixture into LB
4. pSB1AC3 into LB

August 22

• Minimal Media was made to replace what had become soiled after the lab move. Unfortunately, after autoclaving the media, it turned cloudy as there was a suspended precipitate in it. The procedure shall be repeated tomorrow.
• The samples that were seeded yesterday were diluted further to grow in preparation for the midi prep.

August 23

• A miniprep of the seedings from two days ago for the ligation mixture was preformed simply to run a screening digest to test for the presence of the insert. A midi prep of the other seedings was done to further concentrate the DNA.
• A screening digest of the ligation, E0434, I5611 and pSB1AC3 were done with the following enzymes:

1. Ligation construct (I5611 with pSB1AC3)

• AfeI with XbaI (buffer 4 and BSA)
• BSRGI with EcoRI (buffer 2 and BSA)
• PstI (buffer 3 and BSA)

1. E0434

• XmnI with SpeI (buffer 2 with BSA)
• HindIII with PstI (buffer 2 with BSA)
• EcoRI (NEbuffer with no BSA)

1. I5611

• XbaI and PstI (buffer2 and BSA)
• EcoRI and BsrGI (buffer 2 and BSA)
• PstI (buffer 3 and BSA)

1. pSB1AC3

• XmnI and SpeI (buffer 2 with BSA)
• EcoRV with PstI (buffer 3 and BSA)
• EcoRI (NEbuffer with no BSA)

The following ratios of substances were used:

• 1.5uL 10X buffer
• 1.5uL 10X BSA
• 7uL water
• 3uL DNA
• 2uL (one of each) of enzyme