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<h1>Protocol: supported lipid bilayer experiments</h1>
 +
<p>iGEM UCopenhagen 2018</p>
 +
<h2>Lipid film stocks</h2>
 +
<p>Lipid mixtures containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl) is prepared for subsequent use to make supported lipid bilayer and liposomes. This composition is inspired from Chatterjee and colleagues [1]. </p>
 +
<p>The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.</p>
 +
 +
<table>
 +
  <tr>
 +
  <td><strong>Lipid</strong>
 +
  </td>
 +
  <td><strong>MW (g/mol)</strong>
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>DOPC
 +
  </td>
 +
  <td>786.113
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>DOPS
 +
  </td>
 +
  <td>810.025
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>SM
 +
  </td>
 +
  <td>760.2
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Chl
 +
  </td>
 +
  <td>386.7
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Atto655
 +
  </td>
 +
  <td>1366.0
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Biotin DSPE PEG (2000)
 +
  </td>
 +
  <td>3016.0
 +
  </td>
 +
  </tr>
 +
</table>
 +
 +
<p> Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe. </p>
 +
<h3>Materials</h3>
 +
 +
<ul>
 +
<li>25 mg/mL stocks of each lipid* (referred to as "stock 1" in notebook)
 +
<li>Lipid dye Atto655, 0.5 mg/mL (referred to as "stock 1" in notebook)
 +
<li>Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook)
 +
<li>Chloroform and methanol**
 +
</ul>
 +
 +
<p>*Lipids: SM, DOPC, DOPS and Chl. </p>
 +
<p>**A mix of chloroform and methanol is used for DOPC and SM solutions and chloroform alone for the rest of the lipid solutions.</p>
 +
<h3>Procedure</h3>
 +
<p> Dilute 25 mg/mL lipid stocks to new stocks with concentrations of 1 μg/μL (referred to as “stock 2” in notebook).</p>
 +
<p>Dilute biotin and dye to stocks of 0.1 μg/μL and 0.1 μg/μL (referred to as “stock 2” in notebook).</p>
 +
<p>Make lipid mastermixes 1, 2 and 3 from the stocks 2s by adding the following.</p>
 +
 +
 +
<table>
 +
  <tr>
 +
  <td><strong>Lipid</strong>
 +
  </td>
 +
  <td><strong>Mastermix 1</strong>
 +
  </td>
 +
  <td><strong>Mastermix 2</strong>
 +
  </td>
 +
  <td><strong>Mastermix 3</strong>
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>DOPC (1 μg/μL)
 +
  </td>
 +
  <td>75 μL
 +
  </td>
 +
  <td>75 μL
 +
  </td>
 +
  <td>75 μL
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>DOPS (1 μg/μL)
 +
  </td>
 +
  <td>39 μL
 +
  </td>
 +
  <td>39 μL
 +
  </td>
 +
  <td>39 μL
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>SM (1 μg/μL)
 +
  </td>
 +
  <td>133 μL
 +
  </td>
 +
  <td>134 μL
 +
  </td>
 +
  <td>134 μL
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Chl (1 μg/μL)
 +
  </td>
 +
  <td>31 μL
 +
  </td>
 +
  <td>31 μL
 +
  </td>
 +
  <td>31 μL
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Atto655 (0.01 μg/μL)
 +
  </td>
 +
  <td>27 μL
 +
  </td>
 +
  <td>27 μL
 +
  </td>
 +
  <td>-
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>Biotin DSPE PEG (2000) (0.1 μg/μL)
 +
  </td>
 +
  <td>60 μL
 +
  </td>
 +
  <td>-
 +
  </td>
 +
  <td>-
 +
  </td>
 +
  </tr>
 +
</table>
 +
 +
<p>Aliquot mastermixes into 2 μmol portions (10 aliquots). Use brown light-blocking vials for the mixtures containing dye.</p>
 +
<p>Dehydrate lipids by using a nitrogen flow or a ScanVac evaporator for vacuum concentration.</p>
 +
<p>Store lipid film stocks at -20°C.</p>
 +
<h1>Liposomes</h1>
 +
 +
 +
<h2>Materials</h2>
 +
 +
<ul>
 +
 +
<li>Lipid film stocks
 +
<li>LB media</li></ul>
 +
 +
<h2>Procedure</h2>
 +
 +
 +
<p>
 +
Rehydrate lipid stocks and extrude liposomes as described in the <a href="https://static.igem.org/mediawiki/2018/5/5b/T--UCopenhagen--Protocol_Liposome_preparation_calculations_sheet_NH_lab.xlsx">Liposome preparation protocol</a> step 6 to 16. Use LB media to rehydrate lipids.
 +
</p>
 +
<p>
 +
Store at -20<strong>°</strong>C if not used immediately.
 +
</p>
 +
<h1>Confocal microscopy</h1>
 +
 +
 +
<h2>Materials</h2>
 +
 +
<ul>
 +
 +
<li>Liposomes made from lipid mastermix no. 2
 +
<li>LB media
 +
<li>Bacteria (SIEC and SIECΔp1 transformed with GFP gene)</li></ul>
 +
 +
<h2>Procedure</h2>
 +
 +
 +
<p>
 +
Grow bacteria in media for 2.5 hours while inducing expression, 37 oC at 160 rpm*.
 +
</p>
 +
<p>
 +
Prepare a supported lipid bilayer on a microscopy slide as described in the <a href="https://www.chemistry.mcmaster.ca/moran-mirabal/resources/SUPPORTED-LIPID-BILAYER-PREPARATION-SOP-9-2015.pdf">Moran-Mirabel protocol</a> step 8 to 12. Use LB media to wash the slide.
 +
</p>
 +
<p>
 +
Add 100 μL bacteria culture to each slide.
 +
</p>
 +
<p>
 +
Incubate at 37 <strong>°</strong>C for 3 hours.
 +
</p>
 +
<p>
 +
Prepare the confocal fluorescence microscope. Use <em>fluorescence recovery after photobleaching</em> (FRAP) to to investigate if the membrane is intact.
 +
</p>
 +
<p>
 +
Take pictures before and after washing the slide with 100 μL LB media several times. Use 488 nm laser (for GFP) and 635 nm (for Atto655).
 +
</p>
 +
<p>
 +
*Incubation time was inspired by Ruano-Gallego <em>et al. </em>2015 [2].
 +
</p>
 +
<h1>References</h1>
 +
 +
 +
<p>
 +
<strong>[1] </strong>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>
 +
<strong>[2]</strong> Ruano-Gallego, D., Álvarez, B., Fernández, L. A. (2015) Engineering the Controlled Assembly of FIlamentous Injectisomes in E. Coli K-12 for Protein Translocation into Mammalian Cells. ACS Synth. Biol. 4, pp 1030-1041.
 +
</p>
 +
 +
<h1>Overview of SLB protocols</h1>
 +
<p>An overview of the protocols used in the Supported Lipid Bilayers experiments.</p>
 +
<h2><a href="https://static.igem.org/mediawiki/2018/6/6d/T--UCopenhagen--Protocol_Liposome_preparation_step_by_step_NH_lab.pdf">Liposomes preparation step-by-step</a></h2>
 +
<p> Protocol from Nikos Hatzakis’ enzyme lab. Explains the process of making liposomes. When making liposomes in our own lab, we used liquid nitrogen instead of acetone and dry ice.</p>
 +
 +
<h2><a href="https://static.igem.org/mediawiki/2018/5/5b/T--UCopenhagen--Protocol_Liposome_preparation_calculations_sheet_NH_lab.xlsx">Liposome preparation</a></h2>
 +
<p>Excel sheet to calculate amount of lipid added when making liposomes. Goes with the protocol Liposomes preparation step-by-step.</p>
 +
 +
<h2><a href="https://www.chemistry.mcmaster.ca/moran-mirabal/resources/SUPPORTED-LIPID-BILAYER-PREPARATION-SOP-9-2015.pdf">Preparation of supported lipid bilayers</a></h2>
 +
<p>When making supported lipid bilayers it is based on this protocol step 8 to 12.</p>
 +
 +
<h2><a href="https://2018.igem.org/Team:UCopenhagen/Protocols_Lipid_Bilayers">Supported lipid bilayers experiments</a></h2>
 +
<p>Internal protocol for the experiments performed in the confocal microscope with supported lipid bilayers. The protocol contains parts from other external protocols.</p>
 +
 +
 +
 +
 +
 +
  
−
<h1>Supported Lipid Bilayers</h1>
 
 
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Latest revision as of 10:40, 16 October 2018

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Protocol: supported lipid bilayer experiments

iGEM UCopenhagen 2018

Lipid film stocks

Lipid mixtures containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl) is prepared for subsequent use to make supported lipid bilayer and liposomes. This composition is inspired from Chatterjee and colleagues [1].

The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.

Lipid MW (g/mol)
DOPC 786.113
DOPS 810.025
SM 760.2
Chl 386.7
Atto655 1366.0
Biotin DSPE PEG (2000) 3016.0

Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe.

Materials

  • 25 mg/mL stocks of each lipid* (referred to as "stock 1" in notebook)
  • Lipid dye Atto655, 0.5 mg/mL (referred to as "stock 1" in notebook)
  • Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook)
  • Chloroform and methanol**

*Lipids: SM, DOPC, DOPS and Chl.

**A mix of chloroform and methanol is used for DOPC and SM solutions and chloroform alone for the rest of the lipid solutions.

Procedure

Dilute 25 mg/mL lipid stocks to new stocks with concentrations of 1 μg/μL (referred to as “stock 2” in notebook).

Dilute biotin and dye to stocks of 0.1 μg/μL and 0.1 μg/μL (referred to as “stock 2” in notebook).

Make lipid mastermixes 1, 2 and 3 from the stocks 2s by adding the following.

Lipid Mastermix 1 Mastermix 2 Mastermix 3
DOPC (1 μg/μL) 75 μL 75 μL 75 μL
DOPS (1 μg/μL) 39 μL 39 μL 39 μL
SM (1 μg/μL) 133 μL 134 μL 134 μL
Chl (1 μg/μL) 31 μL 31 μL 31 μL
Atto655 (0.01 μg/μL) 27 μL 27 μL -
Biotin DSPE PEG (2000) (0.1 μg/μL) 60 μL - -

Aliquot mastermixes into 2 μmol portions (10 aliquots). Use brown light-blocking vials for the mixtures containing dye.

Dehydrate lipids by using a nitrogen flow or a ScanVac evaporator for vacuum concentration.

Store lipid film stocks at -20°C.

Liposomes

Materials

  • Lipid film stocks
  • LB media

Procedure

Rehydrate lipid stocks and extrude liposomes as described in the Liposome preparation protocol step 6 to 16. Use LB media to rehydrate lipids.

Store at -20°C if not used immediately.

Confocal microscopy

Materials

  • Liposomes made from lipid mastermix no. 2
  • LB media
  • Bacteria (SIEC and SIECΔp1 transformed with GFP gene)

Procedure

Grow bacteria in media for 2.5 hours while inducing expression, 37 oC at 160 rpm*.

Prepare a supported lipid bilayer on a microscopy slide as described in the Moran-Mirabel protocol step 8 to 12. Use LB media to wash the slide.

Add 100 μL bacteria culture to each slide.

Incubate at 37 °C for 3 hours.

Prepare the confocal fluorescence microscope. Use fluorescence recovery after photobleaching (FRAP) to to investigate if the membrane is intact.

Take pictures before and after washing the slide with 100 μL LB media several times. Use 488 nm laser (for GFP) and 635 nm (for Atto655).

*Incubation time was inspired by Ruano-Gallego et al. 2015 [2].

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] Ruano-Gallego, D., Álvarez, B., Fernández, L. A. (2015) Engineering the Controlled Assembly of FIlamentous Injectisomes in E. Coli K-12 for Protein Translocation into Mammalian Cells. ACS Synth. Biol. 4, pp 1030-1041.

Overview of SLB protocols

An overview of the protocols used in the Supported Lipid Bilayers experiments.

Liposomes preparation step-by-step

Protocol from Nikos Hatzakis’ enzyme lab. Explains the process of making liposomes. When making liposomes in our own lab, we used liquid nitrogen instead of acetone and dry ice.

Liposome preparation

Excel sheet to calculate amount of lipid added when making liposomes. Goes with the protocol Liposomes preparation step-by-step.

Preparation of supported lipid bilayers

When making supported lipid bilayers it is based on this protocol step 8 to 12.

Supported lipid bilayers experiments

Internal protocol for the experiments performed in the confocal microscope with supported lipid bilayers. The protocol contains parts from other external protocols.