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Micro-screening apparatus, process, and products

A microcavity and cavity technology, applied in measuring devices, library screening, biochemical equipment and methods, etc., can solve problems such as high cost materials and reagents, difficult instantaneous measurement of kinetic parameters, low throughput, etc.

Inactive Publication Date: 2018-01-02
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Robotic handling systems for assaying protein function in microtiter plates have reduced labor, but are still relatively low-throughput (eg, 100,000 assays per day) and require large quantities of cost-prohibitive materials and reagents
Recently, oil-water emulsion droplets produced in bulk or combined with microfluidic chips have achieved success in high-throughput enzyme engineering applications, however, this technique can be challenging to implement and it is not easy to make kinetic parameters in Instantaneous measurement in real time during the experiment

Method used

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  • Micro-screening apparatus, process, and products
  • Micro-screening apparatus, process, and products

Examples

Experimental program
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Effect test

Embodiment 1

[0247] Example 1: Microarray Single Cell Analysis and Laser Extraction

[0248] Figures 6A-6D depict the general concept and workflow of a microarray of one aspect of the present disclosure (referred to herein as "μSCALE"). Figure 6A shows the platform workflow. 1) A library of protein variants expressed in bacterial or yeast cells is mixed with opaque magnetic beads. 2) Pipette the mixture into the array at a concentration that results in an average single cell occupancy within the microchamber. Various biochemical assays are performed, with or without cell growth, using fluorescence as readout. 3) The array is imaged via fluorescence microscopy. 4) Quantify the fluorescence intensity of each microcavity and use a laser-based extraction method to isolate desired clones from the array, either as single cells or as bulk pools. 5) Culture the extracted cells in liquid or solid medium. 6) Cells are lysed and plasmids are recovered for characterization and / or to generate new ...

Embodiment 2

[0264] Example 2: High Throughput Screening of Protein Binding Interactions

[0265] Axl IgI mock library screening. will encode human Axl Ig1 (amino acid Ala 19 -Pro 131 ) and non-binding Ax1 variants (E59R, T77R) were cloned into the pCT yeast display plasmid between the NheI and BamHI restriction sites (Kariolis, M.S. et al., Nat. Chem. Biol. 1-10 (2014 )). Yeast surface display studies were performed by electroporation of plasmid DNA into Saccharomyces cerevisiae strain EBY100. Soluble Gas6 was recombinantly expressed in human embryonic kidney (HEK) cells using the FreeStyle Max 293Expression System (Invitrogen) and purified as previously described (Kariolis, M.S. et al, Nat. Chem. Biol. 1-10 (2014)). Natural scFv libraries displayed on the surface of yeast were previously described ( variant) (Deventer, J.A. Van & Wittrup, K.D., Yeast Surface Display for Antibody Isolation: Library Construction, Library Screening, and Affinity Maturation. 1131, 151-181 (Humana Pres...

Embodiment 3

[0281] Example 3: Affinity maturation of the Ax1 receptor: error-prone library sorting

[0282] Gas6 / Ax1 was used as a model system to isolate high affinity Ax1 variants that are potent inhibitors of tumor metastasis. The protein engineering strategy involved two iterations of library generation and screening: an initial library of randomly generated Axl mutations and a second library of recombinant Axl mutations derived from variants recovered from the first library.

[0283] A round of MACS using Gas6-coated magnetic beads reduced the theoretical library size to 3.5 × 10 6 Clones were cloned, and arrays were loaded at a concentration that yielded an average of 4 cells per microtube.

[0284] The resulting pool of Ax1 variants was cultured, induced, and incubated with 100 pM Gas6 for 15 hours to allow equilibrium binding. The library was diluted to 12,600 cells / µL to a concentration of an average of 4 cells per 20 µm diameter capillary and imaged with epifluorescence in two...

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Abstract

Microcavity arrays and methods for quantitative biochemical and biophysical analysis of populations of biological variants. Examples include high-throughput analysis of cells and protein products usea range of fluorescent assays, including binding-affinity measurement and time-resolved enzyme assays. Laser-based extraction of microcavity contents. In one aspect, the disclosure is directed to a system including an array comprising a plurality of distinct cavities comprising open first ends and open second ends, an electromagnetic radiation absorbing material associated with cavities, and a pulsed diode laser configured to deliver electromagnetic radiation to the electromagnetic radiation absorbing material. In various embodiment's of the system, the cavities may be fused capillaries.

Description

[0001] related application [0002] This application claims U.S. Provisional Application No. 62 / 119,251 filed February 22, 2015, U.S. Provisional Application No. 62 / 120,803 filed February 25, 2015, 62 filed November 11, 2015 / 250,478 and U.S. Provisional Application No. 62 / 281,545, filed January 21, 2016, each of which is incorporated herein by reference in its entirety. technical field [0003] The present disclosure relates to assaying compounds of interest using microarrays. Background technique [0004] High-throughput measurements have begun to gain insight into the inherent complexity and dense interconnectivity of biological systems. For example, whole-genome sequencing has yielded rich information on key genes and mutations that underlie the pathophysiology of disease, DNA microarrays have allowed careful analysis of transcriptional patterns in various cancers, and large-scale proteomics approaches have Study signaling networks in a growth factor. However, the abi...

Claims

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Application Information

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IPC IPC(8): C40B30/06C12N13/00C12N15/01C40B30/00C40B30/04C40B40/08
CPCC40B30/06G01N33/502G01N33/54366C40B60/12B01J2219/00286B01J2219/00317C40B30/08C12N15/1079G01N33/5304
Inventor T.M.贝尔J.R.科克伦B.陈S.C.奥尔福德S.林A.坎南I.迪莫夫
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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