Difference between revisions of "Team:UCopenhagen/Experiments"
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<h1>Leakiness characterization</h1> | <h1>Leakiness characterization</h1> | ||
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<h1>Egg membrane experiment</h1> | <h1>Egg membrane experiment</h1> | ||
| + | <h1>Experiments with biomimetic membrane</h1> | ||
| + | <h2>1. Introduction</h2> | ||
| + | <p>The membrane is an important component of our protein production system as it separated the bacterial culture from the protein collection chamber. In the final protein production chamber the membrane would be an artificial biomimetic membrane. | ||
| + | In this experiment, we investigated the use of a biomimetic membrane using supported lipid bilayers and liposomes.</p> | ||
| + | <h2>2. Aim</h2> | ||
| + | <p>The first aim of this part of our project was to investigate if the SIEC strain (i.e. the <i>E. coli</i> strain expressing the injectosome) would bind to a biomimetic membrane and secrete | ||
| + | proteins through it.</p> | ||
| + | <p>The second aim was to investigate if the secretion signal, Map20, (biobrick ID) would increase the amount of protein secreted into liposomes.</p> | ||
| + | <h2>3. Membrane composition</h2> | ||
| + | <p>A mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl). The ratio of the membrane components was inspired from Chatterjee and colleagues [1] who showed that EspD, which is required for formation pores in the host membrane, spontaneously bound to lipid vesicles with this lipid composition. This lipid composition is also similar to the composition of the outer leaflet of a mammalian cell plasma membrane [2]. | ||
| + | The dye Atto655 and biotin were also included in the lipid mixtures. Atto655 was included to enable detection of the lipid bilayers and liposome by fluorescence microscopy.</p> | ||
| + | |||
| + | <h1>References</h1> | ||
| + | <p>[1] Chatterjee, A., Caballero-Franco, C., Bakker, D., Totten, S., Jardim, A. (2015) Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD. J Biol Chem. 290 (42) pp. 25579-25594.</p> | ||
| + | <p>[2] Zachowski, A. (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem. J. 294, pp. 1-14.</p> | ||
Revision as of 18:06, 13 October 2018
Cloning
Making BioBricks
Liposome charaterization
Onion charaterization
Leakiness characterization
Egg membrane experiment
Experiments with biomimetic membrane
1. Introduction
The membrane is an important component of our protein production system as it separated the bacterial culture from the protein collection chamber. In the final protein production chamber the membrane would be an artificial biomimetic membrane. In this experiment, we investigated the use of a biomimetic membrane using supported lipid bilayers and liposomes.
2. Aim
The first aim of this part of our project was to investigate if the SIEC strain (i.e. the E. coli strain expressing the injectosome) would bind to a biomimetic membrane and secrete proteins through it.
The second aim was to investigate if the secretion signal, Map20, (biobrick ID) would increase the amount of protein secreted into liposomes.
3. Membrane composition
A mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl). The ratio of the membrane components was inspired from Chatterjee and colleagues [1] who showed that EspD, which is required for formation pores in the host membrane, spontaneously bound to lipid vesicles with this lipid composition. This lipid composition is also similar to the composition of the outer leaflet of a mammalian cell plasma membrane [2]. The dye Atto655 and biotin were also included in the lipid mixtures. Atto655 was included to enable detection of the lipid bilayers and liposome by fluorescence microscopy.
References
[1] Chatterjee, A., Caballero-Franco, C., Bakker, D., Totten, S., Jardim, A. (2015) Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD. J Biol Chem. 290 (42) pp. 25579-25594.
[2] Zachowski, A. (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem. J. 294, pp. 1-14.