Gel-encapsulated microcolony screening

A technology of encapsulation and hydrogel, applied in the determination/inspection of microorganisms, immobilization on/in organic carriers, instruments, etc.

Inactive Publication Date: 2013-12-18
AMYRIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, given the challenge of identifying subtle improvements in metabolic flux from one strain to another in synthetic biology, there remains a need for screening systems and methodologies that are simultaneously more sensitive, reliable, robust, and efficient than existing techniques

Method used

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  • Gel-encapsulated microcolony screening
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Examples

Experimental program
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Effect test

Embodiment 2

[0272] 7.2 Example 2: Encapsulation of cells

[0273] This example describes exemplary methods for encapsulating cells into hydrogels and screening the encapsulated cells for recombinant production of one or more water-immiscible compounds.

[0274] 7.2.1 Fabrication of microfluidic systems

[0275] Microfluidic devices composed of the elastomeric polymer poly(dimethylsiloxane) (PDMS) and containing at least two channels interconnected at T-junctions are prepared using etched wafer substrates (e.g., by photolithography ) as die manufactured.

[0276] Briefly, wafer substrates were prepared by rinsing the wafer with acetone and isopropanol. A photoresist such as SU8-3000 photoresist (Microchem, Newton, MA) is applied to the wafer by spin coating. This photoresist coating is then selectively irradiated with UV light through a mask designed to allow exposure of the photoresist in a selected pattern (e.g., a pattern involving two channels interconnected at T-junctions). Floor....

Embodiment 3

[0288] 7.3 Example 3: Particle Analysis and Sorting

[0289] This example describes exemplary methods for the analysis and sorting of hydrogel particles comprising cells that produce water-immiscible compounds.

[0290] Place a 70 μm BD cell strainer on a 50 ml conical tube. The culture containing the encapsulated cells was applied to the center of the filter with a 5 ml pipette. The particles were washed off the membrane with two 1 ml aliquots of PBS, centrifuged at 100 g for 30 s, resuspended in 1 ml PBS and centrifuged again at 100 g for 30 s. The filtered culture was then added in aliquots to 10 μm Partec filters and centrifuged at 100 g for 90 seconds. Add 1 ml PBS to the filter cake of each filter and resuspend the cake by pipetting up and down. Another 1 ml of PBS was added to the suspension and centrifuged at 100 g for 90 seconds.

[0291] Prepare Nile Red staining solution (2 ml / sample), as needed, by adding 200 µl of Nile Red stock solution (100 µg / ml in EtOH) pe...

Embodiment 4

[0297] 7.4 Example 4: Identification and Selection of Farnesene-Producing Cells

[0298] This example demonstrates the sensitivity and fidelity of detection of heterologous water-immiscible compounds in recombinant yeast strains engineered to produce low to high levels of farnesene. A series of yeast strains produced according to the method described in Example 1 and representing a broad range of farnesene production were encapsulated and sorted according to the methods described in Examples 2 and 3, respectively. Nile red was used as a detection agent. Farnesene levels detected by the skin screening method were compared to levels detected by standard measurements including 2-liter fermentor yield, Nile Red 96-well vibrating plate, and farnesene flux.

[0299] The Nile Red 96-well shaker plate assay was performed as follows. For each strain, pick a single colony from the agar plate into a 1.1 ml 96-well plate containing 360 μl of Bird Seed medium (BSM), 2% sucrose, 0.25N+CRB...

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Abstract

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.

Description

[0001] 1. Cross references to related applications [0002] This application claims U.S. Provisional Patent Application Nos. 61 / 437,214, filed January 28, 2011, and entitled "Gel-Encapsulated Microcolony Screening," and filed May 13, 2011, and entitled "U.S. Priority to U.S. Provisional Application No. 61 / 486,211, which is hereby incorporated by reference in its entirety. 2. Field of invention [0003] The compositions and methods provided herein relate generally to industrial uses of microorganisms. In particular, provided herein are methods and compositions useful for detecting the production of industrially useful compounds such as isoprenoids, polyketides, and fatty acids in cells that have been genetically modified, for example, to produce one or Microbial cells of many of these compounds. 3. Background of the invention [0004] The advent of synthetic biology holds the promise of obtaining microbially produced biofuels, chemicals, and biomaterials from renewable sour...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/50C12P5/00
CPCC12Q1/02G01N33/5005C12P5/007C12P7/02C12N11/04C12N11/10G01N33/50C12P5/00
Inventor J·阿格雷斯蒂
Owner AMYRIS INC
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