Skip to content

Latest commit

 

History

History
217 lines (139 loc) · 8.19 KB

txtl-liposome_water-in-oil.md

File metadata and controls

217 lines (139 loc) · 8.19 KB
Raw

TX-TL Liposome Using Water-in-Oil Emulsion

Note: an updated version of this protocol can be found on the Nucleus container protocols website,

Version 0.1.0, 8 August 2018

Contributors: Aaron Engelhart (University of Minnesota), Jan Gregrowicz (Caltech), Joseph Heili (University of Minnesota), Zoila Jurado (Caltech), Neha Kama (Northwestern), Akshay Maheshwari (Stanford), Richard Murray (Caltech), Milena Popovic (Blue Marble Space), Pasquale Stano (University of Salento), Kazhito Tabata (University of Tokyo), Paola Torre (University of Pennsylvania) Richard Murray (Caltech) Kate Adamala (University of Minnesota)

Overview and Materials

This protocol describes how to create liposomes that contain TX-TL inside of a lipid-based container. The liposomes created by these protocols are between 1 and 10 um in diameter and should be usable with an cell-free protein expression system.

The preparation of liposomes using w/o emulsions as template requires four steps:

Step 0: Deposition of a thin lipid film in a glass vial

Materials:

Step 1: Preparation of the oil containing lipids

Materials:

Step 2: Self-assembly of the Liposomes by centrifugation

Materials:

Step 3: Microscopy Visualization

Materials:

Step 0: Deposition of a thin lipid film in a glass vials

The first step in the protocol is to generate the lipid film required to to form the lipid-in-mineral oil solution. Makes 6 samples with accurate measurement. Each vial will have ~ 15 mg of lipid with 0.1 mol % of 18:1 Lyss-Rho-PE.

  1. Create lipid master mix in a glass beaker. One of two methods may be used:

    • POPC/Lyss-Rhod-PE

      a. Add 4 mL (100 mg) of POPC in chloroform (25 mg/ml)

      b. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL)

      c. Swirl gently until all POPC is dissolved and color is homogeneous.

    • POPC/Cholesterol/Lyss-Rhod-PE

      a. Add 2.668 mL (66.7 mg) of POPC in chloroform (25 mg/ml)

      b. Add 1.332 mL (33.3 mg) of Cholesterol in chloroform (25 mg/ml)

      c. Add 200 uL (0.2 mg) of Lyss-Rho-PE in chloroform (1 mg/mL)

      d. Swirl gently until all POPC is dissolved and color is homogeneous.

  2. Aliquot 700 uL of POPC/Cholesterol/Lyss-Rhod-PE Chloroform solution into 2 mL glass vials (6 vials total)

  3. Place uncapped vials in fume hood, loosely covered with aluminum foil and allow to evacuate overnight in fume hood (~6-8 hours).

    • Note: The purpose of the aluminum foil cover is to protect particles from contaminating the POPC/Cholesterol/Lyss-Rhod-PE as it evaporates.

  4. Move vials to vacuum chamber and vacuum for an additional 2 hours.

  5. Store in -20 degC.

Note: Remaining POPC in chloroform, cholesterol, and Lyss-Rho-PE in chloroform can be stored at -20 degC in the glass vial with PTFE caps and sealed parafilm.

Step 1: Preparation of the oil containing lipids

  1. Mix mineral oil by gentle inversion before use

  2. Place 0.5 mL of mineral oil into each of the vials.

  3. Incubate at 60 degC for 10 min

  4. Vortex for 10 mins

  5. Incubate vials for 3 hrs at 60 degC.

  6. Wrap up the top of the vials with aluminum foil and seal with parafilm.

  7. Sonicate in an heated water bath for 30 minutes,at 60 degC .

    a. Note: If a sonicator is unavailable then repeat 60 degC incubation and vortexing until the lipid is completely dissolved.

Step 2: Self-assembly of the Liposomes by centrifugation

  1. Place 225 uL of centrifuge buffer (100 mM HEPES + 200 mM glucose, pH 8) into a labeled eppendorf tube.

    a. Note: When TX-TL systems are used, the outer solution should also contain the small molecular weight components of the TX-TL system.

  2. Add in 30 uL of inner solution (100 mM HEPES, 200 mM sucrose pH 8, and, if needed, 2 uM of a water-soluble fluorescent dye like HPTS or calcein.) to 500 uL of suspended lipid in oil from Step

    1. Vortex for 30 s. Equilibrate for 10 min at 4 degC.
    • Note: When TX-TL systems are used, buffer is omitted. TX-TL systems come with their own buffers.

  3. Add the emulsion on top of the 225 uL centrifuge buffer by pipetting against the wall of the tube, wait at least 1 minute for the interface to stabilize and flatten between the emulsion and buffer

  4. Centrifuge at 18000 rcf at 4 degC for 15 min

    • Note: If the top phase (oil) is clear, that suggests that droplets have passed into the bottom buffer, becoming vesicles.

  5. Carefully remove as much mineral oil as possible with a gel-loading-pipette-tip from the top.

    • **Note: **The goal is to remove as much oil up to the interface without disturbing the buffer below.

  6. Place 225 uL of wash buffer (100 mM HEPES + 250 mM glucose, pH 8) into a labeled eppendorf tube.

  7. Add the centrifuge buffer (where the vesicles should have formed) to eppendorf tubes with wash buffer. Make sure to use a new tip to avoid contamination.

    • Note: An alternative method is to open the eppendorf tube and use a 21-gauge needle to punch a hole at the bottom of the eppendorf tube. Remove the needle and the close the lid to allow the buffer solution to drip out.

  8. Centrifuge at 12000 rcf at 4 degC for 5 min

  9. Transfer 225 uL from the bottom of the eppendorf tubes with wash buffer into a new labeled eppendorf tube.

Step 3: Microscopy Visualization

  1. Use Frameseal or Spacer (20 mm D x 0.12 mm depth) to make a small chamber on a microscope slide.

  2. Add 10 uL of the final solution to the chamber and seal with cover slip

  3. Wait 5 minutes before imaging (since the liposomes will float everywhere and can be tricky to catch them).

  4. Observe on an inverted microscope. [Sample images] of what you should see:

Alternative method (neha):

  • Use a glass bottom chamber: https://www.thermofisher.com/order/catalog/product/154453

  • Block glass by adding 0.5 mL of a 1mg/mL BSA in PBS solution for 10 minutes (alternatively block by adding 0.5 mL of SuperBlock for 5 min). The blocking step is important because phospholipid vesicles will rupture to some degree on glass.

  • Rinse with PBS 3X. Add 0.5 mL of final buffer from above to chamber. Add 10 uL of vesicle sample to chamber, let settle over a few minutes. Image on scope with green and red channels.

    Acknowledgments:

    This work was possible thank to NSF Research Coordination Network, award number 1901145.