30 August 2007

Construction of pT7-GFP

1. Insert re-digest was run on 1% agarose gel
   • 1. 30 μl insert re-digest
   • 2. 5 μl uncut insert
   • 3. 2 μl 1 kb DNA ladder

Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Electrophoresis| Electrophoresis] in the general protocols page

1. PCR purified vector digest
2. Gel extract purified insert re-digest

Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#DNA Extraction/Purification| DNA Extaction/Purification] in the general protocols page

1. Purified vector and insert were run on 1% agarose gel
   • 1. 6 μl purified insert
   • 2. 3 μl purified vector
   • 3. 2 μl 1 kb DNA ladder

Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Electrophoresis| Electrophoresis] in the general protocols page

1. Ligated vector and insert at 14°C overnight
   • 7 μl purified insert
   • 1 μl purified vector
   • 1 μl T4 ligase
   • 1 μl T4 ligation buffer
2. A negative control with ddH20 instead of insert was also set up

Cell by date - Operating Temperature Range

Construct - pTet-GFP [http://partsregistry.org/Part:BBa_I13522 BBa_I13522]
Temperatures - 37 °C and 20°C Aims: To test for the behaviour of the DNA construct (pTet) at temperatures 20oC and 37oC by observing the amount of fluorescence produced over a period of 24 hours.

• For each temperature two experiments carried out at staggered time points to minimize the time points not measured over night.
• Sampling was initially every 10 minutes however, this was reaccessed and changed to 20 and 30 minute time intervals.
• Measurements will carry on over tomorrow day to give ~30hours of measurements.

Protocol can be found here under Phase 2-Operating Temperature Range on the experimental design page.

Results can here under Results on the experimental design page.

Degradation of GFP

• Tested GFP degradation at 37 °C and 20°C
• The tests were carried out on the same plate as the Operating Temperature Ranges and so the sampling was the same as that for the individual temperatures
• Measurements will carry on over tomorrow day to give ~30hours of measurements

Midiprep of Biobricks

1. Midipreped sample left overnight in isopropanol was washed with 70% ethanol
   Previous protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Midiprep| Midiprep] in the general protocols page
2. Solution was dissolved in 300 µl of ddH₂O
3. Resulting concentration of DNA was 40 ng/µl (lower than expected!)

Vesicles

Results Samples 1 through 5, prepared the day before were collected for observation under the microscope. The following results were obtained:

• Vesicles were found in sample 1.
• A single vesicle was found in sample 2.
• No vesicles were found in samples 3-5, in spite of the presence of many GFP aggregates.

Vesicles found in Sample 1. Left: Several tiny vesicles - and one large - under white light. Centre: The same objects under fluorescence. Right: The same picture as in the centre, but with gamma correction and full saturation. Note that only the large vesicle shows good fluorescence. Two of the brighter grey spots in the enhanced image correspond to small vesicle-like objects, but they are also surrounded by fainter grey spots. The grey colour comes from a very faint fluorescence being enhanced by gamma correction. Because it is very faint, it does not show as green. Also partly visible in the white light picture is the 'streaking' effect. When the slide cover gets placed over the microscope slide, it may sometimes get pushed around. This moves the objects underneath it, sometimes smearing vesicles across a millimetre or so. This forms a sort of trail of progressively larger vesicles, sometimes all fluorescent. Occasionally, the final large vesicle can be seen partly ruptured. It is not shown well in the picture to the left, but the large vesicle is actually the end of one such streak, with its smaller parts trailing off towards the top-left of the picture.

Vesicles found in Sample 1. Left: Several large and small vesicles under white light. Centre: The same objects under fluorescence. Right: The same picture as in the centre, but with gamma correction and full saturation. The grey spots in the enhanced image correspond to fluorescent vesicles. The grey colour comes from a very faint fluorescence being enhanced by gamma correction. Because it is very faint, it does not show as green in the picture. Although the camera did not capture the fluorescent emission, it was in fact visible in the microscope.

Single 2μm vesicle found in Sample 2. Left: Near the centre of the picture, the vesicle under white light. Centre: The same objects under fluorescence. Right: The same picture as in the centre, but with gamma correction and full saturation. This was the only vesicle found in Sample 2, in spite of many fluorescent artefacts being present.