A method to homogenize the concentration of beads across multiple liquid volumes
AssigneeNuclera Ltd
InventorsZachary GABER, Sumit KALSI, Johnathon LILLEY, Andreas Michael WAEBER
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Abstract
The invention provided herein relates to methods for normalising the concentration of beads in droplets on a microfluidic device, and methods for cell-free protein synthesis and characterisation of the expressed proteins.
Claims24
A method for creating aqueous droplets having homogeneous concentrations of suspended beads, wherein the method comprises:
- a. taking a reservoir containing an aqueous liquid having beads suspended therein,
- b. forming multiple droplet sub-volumes from the reservoir,
- c. merging two or more of the droplets,
- d. splitting the merged droplets,
- e. rearranging the set of droplets, and
- f. repeating steps c., d. and e. one or more times such that each of the final split droplets contains a homogeneous concentration of suspended beads.
The method according to any one preceding claim, wherein the beads are greater than 10 μm in diameter.
The method according to any one preceding claim, wherein the beads average 25 μm in diameter.
The method according to any one preceding claim wherein, the homogeneous bead droplets are merged with droplets having substances which bind to the beads.
The method according to any one preceding claim wherein the beads are used to bind to proteins.
A method for synthesising, characterising and purifying one or more proteins having detection tags and binding tags, the method comprising the steps of:
- i mixing a cell-free protein expression system with one or more nucleic acid templates to form a plurality of merged liquid volumes;
- either
- iia splitting the merged volumes into at least two aliquots and allowing protein expression in at least two aliquots;
- or
- iib allowing protein expression then splitting into at least two aliquots;
- iii adding a detector reagent which binds to the detection tags to at least one of the aliquots in order to determine the level of protein expression and determining the conditions where protein is expressed in soluble form;
- iv selecting the volumes where protein has been solubly expressed and adding magnetic beads to a sub-population of the aliquots, wherein the beads bind to the binding tags and the beads in the droplets have been merged and split one or more times such that each of the final split droplets contains a homogeneous concentration of suspended beads;
- v separating the magnetic beads from the expression reagents;
- vi optionally adding wash reagents to the magnetic beads to remove unbound material;
- vii eluting the bound protein from the magnetic beads by disrupting the binding to the binding tag;
- and
- viii adding a detector reagent which binds to the detection tags to at least one of the eluted aliquots in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
The method according to any one preceding claim comprising expressing one or more proteins in one or more droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes, wherein the proteins have a ccGFP11 peptide amino sequence tag and a Strep-tag, splitting the droplets into at least two volumes, merging droplets containing ccGFP1-10 into one of the droplets following the expression process and measuring the uniformity of the fluorescent signal to determine the degree of aggregation of the expressed protein, taking corresponding droplets without ccGFP1-10 which contain protein having been solubly expressed and adding magnetic beads which bind to the strep-tag, wherein the droplets having the magnetic beads have been merged and split one or more times such that each of the final split droplets contains a homogeneous concentration of suspended beads, immobilizing and washing the beads to remove unbound material;
- eluting the POI by disrupting the strep binding, adding droplets containing ccGFP1-10 following the elution process and measuring the fluorescent signal in order to determine the level of protein purification and determining the conditions where purified soluble protein is obtained.
The method according to any one preceding claim, wherein at least 8 different nucleic acid templates are screened against at least 4 different expression reagents on the same device.
The method according to any one preceding claim, wherein the device can separately manipulate at least 32 droplets containing beads.
The method according to any one preceding claim, wherein the final dispensed homogenised concentration of the beads is 20% v/v+/−10% of each droplet.
Dependencies are inferred from the claim text.