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TX-TL Liposome Protocol

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

Step 1: Liposome-oil emulsion

  1. Mix mineral oil by gentle inversion before use (Mineral oil can dissolve lipid better than paraffin)
  2. Place 0.5 mL of mineral oil in the vials
  3. Vortex for 10 mins
  4. Incubate vials at 50C for 10mins
  5. Repeat 50C incubation and vortexing until the lipid is completely dissolved.

Notes:

  • After 3 rounds of this I put the vials to incubate at 60C for 3.5 hours then vortexed it, the mixture looks a little different than before. The consistency looked soft.

  • 28 March 2018: What helps in later trials was leaving it in heated @ 60C sonicator bath for 1 hr.

Step 2: Making liposomes

  1. Place 20 μL of centrifuge buffer (PBS + 300 mM sucrose, pH 8) into a labeled eppendorf tube

  2. Add 2 μL of reaction into 300 μL phospholipid solution, vortex for 30 s to create an emulsion

  3. Add the emulsion on top of the 20 μL centrifuge buffer, wait for the interface to stabilize and flatten between the emulsion and buffer

  4. Centrifuge at 11000 RPM for 30 s (tabletop centrifuge works well)

  5. Carefully remove the mineral oil, taking 10 μL of the centrifuge buffer (where the vesicles have been formed). Make sure to use a new tip to avoid contamination.

  6. Add 10 μL of solution (where vesicles hopefully "live") to 10 μL of final buffer (50 mM HEPES + 300 mM sucrose, pH 8), careful not to touch the sides of tube

  7. Mix gently, pipetting up and down with a new tip"

Step 3: Microscopy

  1. Use Frameseal to make a small chamber on a coverslip

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

  3. Observe on microscope. Sample images of what you should see:

Sample microscope images

Credits

This protocol builds on vesicle protocols by Vincent Noireaux [1] and Kate Adamaala [2], with modifications by Zoila Jurado and Bruce Whitman.

Additional thanks to Malin Jonsson, Kate Adamala, and Vincent Noireaux

Full list of contributors

  • Zoila Jurado: creation and documentation of initial protocol
  • Richard Murray: conversion of protocol to MarkDown from ipynb

References

Milena Popovic