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

=In-Veso Gene Expression: Implementation=

 Introduction Specifications Design Modelling Implementation Testing/Validation Notes References </ul> <br style="clear:both">

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1.1 Forming Vesicles
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. Aims: <span class="_toggler_toggle-item1-1-1">[+] Constant Conditions
 * 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
 * 125μl phospholipid added to mineral oil solvent
 * Strict adherence to protocol (except for interface (feeding) solution)

<span class="_toggler_toggle-item1-1-2">[+] Variables
 * Number of vesicles
 * Size of vesicles
 * Modification of protocol at interface stage
 * Life span of chassis

<span class="_toggler_toggle-item1-1-3">[+] Sampling
 * 2 microscopic slides
 * 100x light microscope

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1.2 Adding GFP into Vesicles
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: <span class="_toggler_toggle-item1-2-1">[+] Constant Conditions
 * To compensate for osmolarity and form vesicles with GFP inside
 * Visualize them under fluorescence microscope
 * Note the range of size of vesicles and lifespan
 * 125μl phospholipid added to mineral oil solvent
 * Strict adherence to protocol (except for interface (feeding) solution)

<span class="_toggler_toggle-item1-2-2">[+] Variables
 * Number of vesicles
 * Size of vesicles
 * Modification of protocol at interface stage
 * Life span of chassis

<span class="_toggler_toggle-item1-2-3">[+] Sampling
 * 2 microscopic slides
 * 100x flurescence microscope

<span class="_toggler_toggle-item1-2-4">[+] Controls
 * 100x GFP standard solution

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
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: <span class="_toggler_toggle-item1-3-1">[+] Constant Conditions
 * 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
 * 0.05mg/ml phospholipid suspended oil.

<span class="_toggler_toggle-item1-3-2">[+] Variables
 * Number of vesicles
 * Size of vesicles
 * Modification of protocol at interface stage
 * Modification of protocol suspension volume
 * Modification of enclosed solution (using cell extract)
 * Modification of external solution (for compensation of osmotic pressure)
 * Life span of chassis

<span class="_toggler_toggle-item1-3-3">[+] Sampling
 * 2 microscopic slides
 * 100x flurescence microscope, with oil immersion and phase contrast

<span class="_toggler_toggle-item1-3-4">[+] Controls
 * S30 cell extract
 * 100x diluted GFP solution
 * Comparison to previous 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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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):

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/15 view=threemonths format=%name/%year-%month-%day weekstart=7