IGEM:IMPERIAL/2007/Notebook/2007-9-11

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/11 view=threemonths format=%name/%year-%month-%day weekstart=7

LuxR Purification


The gel suggests that fractions 15-18 contain the LuxR protein.

Plate Evaporation
We have been investigating how to prevent evaporation from our samples being measured in a plate reader. Because of the length of our testing, evaporation is a big problem. We are looking at use of organic oils to prevent evaporation. Today we tested to see if GFP is still fluorescence in oil. From looking in the literature we tested two types of oil at three volumes; The results are on the following link to the Results
 * Parafin Oil - 10ul, 20ul, 90ul
 * Mineral Oil - 10ul, 20ul, 90ul

Formation of Vesicles
Three commercial S30 cell extract mixes were prepared, with the ratio of 1:4:3 AA Mixture : Premix : S30 Extract. These were then used to form three reactions:
 * 50&mu;l Negative Control: 33,3&mu;l of S30 mix, 16,7&mu;l nuclease free water.
 * 50&mu;l Experiment: 33,3&mu;l of S30 mix, 3,8&mu;l Plux GFP DNA, 12,9&mu;l nuclease free water.
 * 20&mu;l Positive Control: 13,4&mu;l of S30 mix, 1,5&mu;l Plux GFP DNA, 5,1&mu;l nuclease free water.

Two of the three suspensions prepared the day before were used to prepare emulsions containing commercial S30 cell extract mixes:


 * Experiment: 10ml emulsion with 50&mu;l of S30 cell extract with Plux GFP DNA
 * Negative Control: 10ml emulsion with 50&mu;l of S30 cell extract without DNA.

The third suspension was used to prepare four interfaces (2ml suspension over 3ml Solution A), to become four samples:
 * Sample 1: Experiment, with circulation by syringe
 * Sample 2: Experiment, without circulation by syringe
 * Sample 3: Negative Control, with circulation by syringe
 * Sample 4: Negative Control, without circulation by syringe

The remaining 20&mu;l of S30/DNA reaction was used to form postitive controls:
 * Concentrated Positive Control: 15&mu;l of S30/DNA reaction
 * Diluted Positive Control: 5&mu;l of S30/DNA reaction diluted into 295&mu;l of Solution A

AHL was then added to each of the four samples and the two positive controls above, to reach a concentration of 5&mu;M/dm3.

Results
Samples 1-4 were looked at, as well as both emulsions before and after centrifugation, and the two positive controls.


 * Emulsion - Experiment (No pictures taken)
 * Before CF: Vesicles of around 2&mu;m diameter found, plus many artifacts.
 * After CF: Very few vesicles found, plus many artifacts.
 * Emulsion - Positive Control (No pictures taken)
 * Before CF: Vesicles of around 2&mu;m diameter found, plus many artifacts.
 * After CF: Very few vesicles found, plus many artifacts.
 * Positive Controls
 * Concentrated: Faint fluorescence throughout, and some aggregates.
 * Diluted: Tiny bright specs of green fluorescence, plus some large aggregates. (See pictures below.)
 * Sample 1 (Experiment): Very few vesicles found, with faint fluorescence inside. Many fluorescent aggregates.
 * Sample 2 (Experiment): Vesicles found, and one with very strong green fluorescence. All other vesicles with faint green fluorescence.
 * Sample 3 (Negative Control): Few vesicles found, but with no fluorescence inside. Large fluorescent object found, and possibly contaminated.
 * Sample 4 (Negative Control): Many vesicles found, but with no fluorescence inside. Bacterial contamination present. Strange textures found that may be oil from the collection process. (No images.)

Cloning of Biobricks

 * 1) Placed 5 ml of LB in a 15 ml tube
 * 2) Added appropriate antibiotics into the tube
 * 3) Picked 6 colony from the fresh overnight plate
 * 4) Innoculated the colony in the LB media
 * 5) Incubated overnight at 37 °C
 * 6) *6x1 Biobricks cloned

Maxiprep of Biobricks (preparation)

 * 1) Cells were spun down and pelleted
 * 2) Cells stored at -20 °C for Maxiprep tomorrow