22 August 2007

Midiprep of Biobricks

Midipreped 6 Biobricks

• 2. BBa_I13522 [ptet-GFP]
• 21. BBa_J23039 [ptet-luxI]
• 23. BBa_I13504 [GFP]
• 24. BBa_R0062 [pLux (no luxR)]
• 25. BBa_J37032 [pLux-GFP]
• 26. BBa_S03119 [tet-LuxR]

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

Miniprep of Biobrick

Minipreped 2x1 Biobrick

• 14. BBa_R0051 [pcI]

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

Restriction Digest of Biobricks

1. 4 DNA samples were digested by EcoRI and PstI
   • Samples 2, 14a, 14b, 24
2. 4 DNA samples were digested by Xbai
   • Samples 21, 23, 25, 26
3. 4 μl of DNA was digested by 1 μl of enzymes at 37°C for 1 hour

Agarose Gel Electrophoresis of Biobricks

1. Checked 7 Biobricks and 7 Digests on 1% agarose gel

Protocols can be found at 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

Conclusions:

• Part 14 is confirmed to be faulty
• Part 21 does not have the right size

In Vivo Testing of pT7 (Continued)

Continue testing pT7 in vivo at 37^oC. This test was started on 21-08-2007 and has been left over night.

Protocol can be found here under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_1.1 Phase 1-In vivo testing] on the experimental design page.

Results can here under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Results_2.1 Results] on the experimental design page

In Vitro Testing of pTet 37^oC (Continued)

Continued with the testing of pTet in vitro which was started on 21-08-2007 at 11.30pm. This experiment will carry on until fluorescence reaches that of the control.

Protocol can be found here under Phase 1-In vitro testing on the experimental design page.

Results can here under Results on the experimental design page

In vitro Testing of pT7 37^oC

Initial testing of pT7 in vitro at 37^oC. This experiment will carry on until fluorescence reaches that of the control.

Protocol can be found here under Phase 1-In vitro testing on the experimental design page.

Results can here under Results on the experimental design page

Operating Range of in vitro Testing of pTet

Initial testing of operating ranges of pTet in vitro at 10^oC and 45^oC. The sampling of this was every 30minutes.

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

Results can here under ... on the experimental design page

Vesicles Formation with GFP

Microscope pictures from sample 4 (2ml emulsion, centrifuge for 20min at 30x g). Left: A fluorescent object. Right: The same object under white light.
Strange structures. Left: Aggregates in the oil-lipid emulsion. Right: Air-water interface and an unexpected 'bubbly' structure in the aqueous region.

Formation of Vesicles

Using a prepared well-dessicated, well sonicated DOPC/mineral oil suspension from yesterday,

• 2ml of suspension was taken to prepare an interface according to protocol.
• 10x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
• Special care was taken to protect the GFP solution from light at all times.

4 samples prepared:

• Sample 1: 2ml of emulsion, no centrifuge.
• Sample 2: Following the protocol, with 2ml suspension interface and 100μl of emulsion added.
• Sample 3: 2ml of emulsion, centrifuge at 120x g for 10mins.
• Sample 4: 2ml of emulsion, centrifuge at 30x g for 20mins.

Results

Light Microscopy (Magnification - 100x):

• Emulsion: Vesicles seen, presumably monolayer.
• Sample 1: Water-oil interface observed. Presumably the phospholipids have clumped altogether.
• Sample 2: Vesicles seen.
• Sample 3: Vesicles seen.
• Sample 4: Vesicles seen.

Fluorescence Microscopy (Magnification - 100x):

• Emulsion: GFP vesicles seen. Lots of GFP artifacts.
• Sample 1: No fluorescent vesicles observed.
• Sample 2: No fluorescent vesicles observed.
• Sample 3: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artifacts.
• Sample 4: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artifacts.

Preparations

No preparations were carried out.