GEL-ENCAPSULATED MICROCOLONY SCREENING
Provided herein are methods and compositions useful for detecting the production of industrially useful compounds (e.g., isoprenoids, polyketides, and fatty acids) in a cell, for example, a microbial cell genetically modified to produce one or more such compounds. In some embodiments, the methods comprise encapsulating the cell in a hydrogel particle, and detecting the compound within the hydrogel particle.
1 . A method of detecting recombinantly produced water-immiscible compound in a cell, the method comprising:
(a) encapsulating the cell in a hydrogel particle; and
(b) detecting the recombinantly produced water-immiscible compound within the hydrogel particle.
2 . The method of claim 1 , wherein said detecting comprises contacting the hydrogel particle with a fluorescent dye that directly binds to the recombinantly produced water-immiscible compound and detecting the fluorescent dye within the hydrogel particle.
3 . The method of claim 2 , wherein the fluorescent dye is a solvatochromic dye.
4 . The method of claim 2 , wherein the fluorescent dye is Nile Red.
5 . The method of claim 1 , wherein said detecting comprises normalizing the amount of water-immiscible compound within the hydrogel particle to the amount of biomass within the hydrogel particle.
6 . The method of claim 1 , wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, and a plant cell.
7 . The method of claim 5 , wherein the cell is a yeast cell.
8 . The method of claim 1 , wherein the recombinantly produced water-immiscible compound is selected from the group consisting of an isoprenoid, a polyketide and a fatty acid.
9 . The method of claim 1 , wherein the hydrogel particle is capable of retaining a water-immiscible compound.
10 . The method of claim 1 , wherein the hydrogel comprises agarose.
11 . The method of claim 1 , wherein the hydrogel particle is less than about 1 millimeter in diameter.
12 . The method of claim 1 , wherein the hydrogel particle is less than about 100 micrometers in diameter.
13 . The method of claim 1 , wherein the hydrogel particle is less than about 50 micrometers in diameter.
14 . The method of claim 1 , wherein the hydrogel particle is about 50, 45, 40, 35, 30, or 25 micrometers in diameter.
15 . The method of claim 1 , wherein said encapsulating comprises contacting the cell with an aqueous hydrogel suspension under conditions sufficient to form a hydrogel particle comprising the cell.
16 . The method of claim 15 , wherein said conditions comprise contacting the aqueous hydrogel suspension comprising the cell with a fluorocarbon oil comprising a fluorosurfactant.
17 . The method of claim 16 , wherein said contacting with a fluorocarbon oil comprises loading the aqueous hydrogel suspension comprising the cell onto a microfluidic device comprising the fluorocarbon oil, wherein said hydrogel suspension contacts the fluorocarbon oil at a T-junction of the microfluidic device, wherein said contacting with the fluorocarbon oil results in formation of a non-aqueous hydrogel particle comprising the cell.
18 . The method of claim 17 , further comprising the step of separating the hydrogel particle from the fluorocarbon oil.
19 . The method of claim 1 , further comprising the step of culturing the cell within the hydrogel particle prior to said detecting.
20 . The method of claim 19 , wherein said culturing is for a period of 12 to 24 hours.
21 . The method of claim 1 , wherein the recombinantly produced water-immiscible compound is a terpene, C 5 isoprenoid, C 10 isoprenoid or C 15 isoprenoid.
22 . The method of claim 21 , wherein the recombinantly produced water-immiscible compound is farnesene.
23 . A method of detecting recombinantly produced water-immiscible compound in a cell, the method comprising:
(a) contacting the cell with an aqueous hydrogel suspension;
(b) loading the aqueous hydrogel suspension comprising the cell onto a microfluidic device comprising a fluorocarbon oil, wherein said hydrogel suspension contacts the fluorocarbon oil at a T-junction of the microfluidic device, wherein said contacting with the fluorocarbon oil results in formation of a non-aqueous hydrogel particle comprising the cell;
(c) separating the hydrogel particle from the fluorocarbon oil;
(d) culturing the cell within the hydrogel particle;
(e) contacting the hydrogel particle with a fluorescent dye that directly binds to the recombinantly produced water-immiscible compound; and
(f) detecting the fluorescent dye within the hydrogel particle.
24 . A method of screening a library of cells for a cell recombinantly producing a water-immiscible compound, the method comprising:
(a) encapsulating each cell of the library in a hydrogel particle;
(b) detecting recombinantly produced water-immiscible compound within each hydrogel particle; and
(c) selecting a cell producing said recombinantly produced water-immiscible compound.
25 . A method of enriching a population of cells for cells recombinantly producing a water-immiscible compound, the method comprising:
(a) providing a population of hydrogel particles, wherein the population comprises hydrogel particles that encapsulate a cell or a clonal population of cells genetically modified to produce a water-immiscible compound;
(b) detecting a hydrogel particle comprising recombinantly produced water-immiscible compound;
(c) recovering the cell or clonal population of cells from the hydrogel particle of step (b);
(d) re-encapsulating the cell or clonal population of cells from step (c); and
(e) repeating steps (a)-(c).
26 . A method of encapsulating a cell within a hydrogel particle, the method comprising:
(a) contacting the cell with an aqueous hydrogel suspension; and
(b) loading the aqueous hydrogel suspension comprising the cell onto a microfluidic device comprising a fluorocarbon oil, wherein said hydrogel suspension contacts the fluorocarbon oil at a T-junction of the microfluidic device, wherein said contacting with the fluorocarbon oil results in formation of a non-aqueous hydrogel particle comprising the cell.
27 . A hydrogel-encapsulated cell or clonal cell population comprising recombinantly produced water-immiscible compound.
28 . The hydrogel-encapsulated cell or clonal cell population of claim 27 contacted with a fluorescent solvatochromic dye.
29 . The hydrogel-encapsulated cell or clonal cell population of claim 27 contacted with Nile Red.
30 . The hydrogel-encapsulated cell or clonal cell population of claim 27 , wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, and a plant cell.
31 . The hydrogel-encapsulated cell or clonal cell population of claim 27 , wherein the cell is a yeast cell.
32 . The hydrogel-encapsulated cell or clonal cell population of claim 27 , wherein the recombinantly produced water-immiscible compound is selected from the group consisting of an isoprenoid, a polyketide and a fatty acid.
33 . The hydrogel-encapsulated cell or clonal cell population of claim 32 , wherein the recombinantly produced water-immiscible compound is a terpene, C 5 isoprenoid, C 10 isoprenoid or C 15 isoprenoid.
34 . The hydrogel-encapsulated cell or clonal cell population of claim 32 , wherein the recombinantly produced water-immiscible compound is farnesene.
35 . A hydrogel particle comprising a cell or clonal cell population, and further comprising recombinantly produced water-immiscible compound.
36 . The hydrogel particle of claim 35 contacted with a fluorescent solvatochromic dye.
37 . The hydrogel particle of claim 35 contacted with Nile Red.
38 . The hydrogel particle of claim 35 , wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, and a plant cell.
39 . The hydrogel particle of claim 37 , wherein the cell is a yeast cell.
40 . The hydrogel particle of claim 35 , wherein the recombinantly produced water-immiscible compound is selected from the group consisting of an isoprenoid, a polyketide and a fatty acid.
41 . The hydrogel particle of claim 40 , wherein the isoprenoid is a terpene, C 5 isoprenoid, C 10 isoprenoid or C 15 isoprenoid.
42 . The hydrogel particle of claim 40 , wherein the isoprenoid is farnesene.
43 . The hydrogel particle of claim 42 , wherein the recombinant yeast cell comprises a nucleic acid encoding farnesene synthase.
44 . The hydrogel particle of claim 35 , wherein the hydrogel particle is capable of retaining a water-immiscible compound.
45 . The hydrogel particle of claim 44 , wherein the water-immiscible compound is selected from the group consisting of an isoprenoid, a polyketide and a fatty acid.
46 . The hydrogel particle of claim 44 , wherein the water-immiscible compound is farnesene.
47 . The hydrogel particle of claim 35 , wherein the hydrogel comprises agarose.
48 . The hydrogel particle of claim 35 , wherein the hydrogel particle is less than about 1 millimeter in diameter.
49 . The hydrogel particle of claim 35 , wherein the hydrogel particle is less than about 100 micrometers in diameter.
50 . The hydrogel particle of claim 35 , wherein the hydrogel particle is less than about 50 micrometers in diameter.
51 . The hydrogel particle of claim 35 , wherein the hydrogel particle is about 50, 45, 40, 35, 30, or 25 micrometers in diameter.