IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation

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In-Veso Gene Expression: Implementation



Contents



Phase 1. Vesicle Formation


1.1 Forming Vesicles

Overall Status
Results available
Construct Status
DOPC / Mineral Oil Prepped - 14/08/07
DOPC / Mineral Oil We have vesicles! - 15/08/07


Constructs:

  • DOPC (1,2-Oleoyl-sn-Glycero-3-phosphocholine) / Mineral Oil
  • POPC (1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine) / Dodecane / Span 80

Based on our specifications, we have decided to use the 'Mineral Oil' method to build our in-veso chassis. This will allow us greater flexibility with the size of vesicles formed, as with the number of vesicles we can produce. We have also decided on using different materials to construct our vesicles - one that is recommended by researchers at Imperial College, and the other that is most frequently used in literature.

Protocol
Results

Aims:

  • To form vesicles
  • Visualize them under light microscope and be able to differentiate them from air bubbles
  • Note the range of size of vesicles and lifespan


[+] Constant Conditions

[+] Variables

[+] Sampling

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1.2 Adding GFP into Vesicles

Overall Status
Results Available
Construct Status
DOPC / Mineral Oil Prepped - 15/08/07
DOPC / Mineral Oil No fluorescence - 16/08/07
DOPC / Mineral Oil Fluorescence at membrane - 17/08/07
DOPC / Mineral Oil Inconclusive - 20/08/07
DOPC / Mineral Oil Glowing vesicles, but still inconclusive - 22/08/07
POPC / Dodecane Glowing vesicles, but still inconclusive - 24/08/07
DOPC / Dodecane (No overnight) Vesicles oserved
03/09/2007
POPC / Dodecane (No overnight) Vesicles observed
03/09/2007


Constructs:

  • DOPC (1,2-Oleoyl-sn-Glycero-3-phosphocholine) / Mineral Oil
  • POPC (1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine) / Dodecane / Span 80

The next step in building our chassis is to ensure that we are able to control what we want inside our chassis. As a means of proof of feasibility, diluted GFP standard solution was added to the emulsion instead, and the results seen using fluorescence mircrosopy.


Aims:

  • To compensate for osmolarity and form vesicles with GFP inside
  • Visualize them under fluorescence microscope
  • Note the range of size of vesicles and lifespan


[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls


Protocol
Results


Main Conclusions:

  • GFP was successfully enclosed inside vesicles.
  • It is possible to make vesicles with GFP without the overnight incubation step.
  • It is possible to make vesicles with GFP with 10ml instead of 50ml of lipid-oil suspension.
  • Dodecane seems to work better than mineral oil with both DOPC and POPC.

Outstanding Questions

  • It is still not clear whether the external GFP aggregates entered the solution encapsulated in vesicles or not. Hence, there is no distinction between encapsulation efficiency and stability. Control experiments should be carried out.
  • The emulsions produced are still not very stable - they tend to coalesce and precipitate fairly quickly. This is probably due to the low adsorption rate of phospholipids. There are two possible solutions - either adding Span-80, which has a higher adsorption rate and better stabilising properties, or stir the emulsion for a longer period at a slower rate (giving more time for phospholipid adsorption to occur).
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1.3 Adding Cell Extract into Vesicles

Overall Status
Results Available
Construct Status
POPC / Dodecane
Noireaux protocol
No vesicles found.
04/09/2007
POPC / Dodecane
50ml suspension
Vesicles produced. Very good results
04/09/2007
DOPC / Dodecane
10ml suspension
Vesicles only without interface prepared.
04/09/2007
POPC / Dodecane
10ml suspension
Vesicles both with and without interface.
04/09/2007
POPC / Dodecane
10ml suspension
50μl Promega S30
NO DNA
Vesicles found, but no fluorescence. Results as expected.
11/09/2007
POPC / Dodecane
10ml suspension
50μl Promega S30
Plux GFP Construct
GFP EXPRESSED inside only one vesicle. Results in other vesicles are inconclusive.
11/09/2007


Constructs:

  • DOPC (1,2-Oleoyl-sn-Glycero-3-phosphocholine) / Dodecane
  • POPC (1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine) / Dodecane
  • DOPC / Span80 / Dodecane
  • POPC / Span80 / Dodecane
  • POPC / DOPC / Span80 / Dodecane
  • POPC / DOPC / Dodecane


Aims:

  • To compensate for osmolarity and form vesicles with Cell Extract inside
  • Visualize them under fluorescence microscope
  • Note the range of size of vesicles and lifespan


[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls




Protocol
Results


Main Conclusions:

Outstanding Questions

  • Osmotic pressure is still an issue that has not been addressed properly. Calculations and hypotheses should be made and tested through experiment. Osmotic pressure affects the stability of vesicles, and therefore a better understanding of this aspect would help explain why components that are expected to be found inside vesicles are being found outside.
  • Gene expression inside vesicles has not yet been tested. Control experiments must be designed in order to properly verify the reaching of this milestone.
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Phase 2. Vesicle Permeabilisation

Phase 3. Process Optimisation and Reproducibility

Phase 4. Application Experiments

Lab Notebook

  • Week 5 (6 Aug): Pilot Experimentation of Vesicles
  • Week 6 (13 Aug): Pilot Experimentation of Vesicles
  • Week 7 (20 Aug): Pilot Experimentation of Vesicles
  • Week 8 (27 Aug): Enclosing GFP inside Vesicles
  • Week 9 (3 Sep): Enclosing cell extract inside Vesicles
  • Week 10 (10 Sep):


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