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 [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.1 Phase 1-In vitro 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 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 [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.1 Phase 1-In vitro testing] on the experimental design page.

• Results can be found under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Results_2.1 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 [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase2/Protocol_2.1.1 Phase 2-Operating Temperature Ranges] 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.