Heterologous expression libraries were prepared as previously described23 (link). In brief, genomic DNA was extracted from overnight cultures of the source bacterial strain24 (link). DNA was sheared to 2–8 kb by focused ultrasonication (Covaris E220 with miniTUBE red) and fragments were cloned into PCR-linearized expression vector pZE21 (primer #1–2) by blunt-ended ligation (Epicentre FastLinkTM kit). Before transformation, the ligation products were separated on a 0.5% agarose gel, the region between 5 and 10 kb was excised and DNA was extracted using a gel extraction kit (Qiagen). Ligation products were transformed into E. cloni®10G Elite competent cells (Lucigen) by electroporation. Overnight grown colonies were picked and arrayed in 384-well format into liquid LB medium supplemented with kanamycin using a colony picking robot (Molecular Devices QPix 420). After incubation overnight at 37°C, plates were replicated in duplicate onto LB agar plates supplemented with kanamycin. The first plate served for the initial drug assay described below to identify plates that contain drug-metabolizing gain-of-function hits. The second plate was stored at 4°C for use in the secondary assay to localize drug metabolizing clones within active plates as described below. Primer #3–4 were used for Sanger sequencing of representative library clones and identified drug-metabolizing clones.
Qpix 420
Manufactured by Molecular Devices
The QPix 420 is a colony picking and imaging system designed for automated colony picking and imaging of bacterial and yeast colonies. It features a high-resolution camera for colony imaging and a robotic arm for precise colony picking.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using qpix 420
1
Heterologous Expression Library Construction
2
Heterologous Expression Library Construction
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Heterologous expression libraries were prepared as previously described23 (link). In brief, genomic DNA was extracted from overnight cultures of the source bacterial strain24 (link). DNA was sheared to 2–8 kb by focused ultrasonication (Covaris E220 with miniTUBE red) and fragments were cloned into PCR-linearized expression vector pZE21 (primer #1–2) by blunt-ended ligation (Epicentre FastLinkTM kit). Before transformation, the ligation products were separated on a 0.5% agarose gel, the region between 5 and 10 kb was excised and DNA was extracted using a gel extraction kit (Qiagen). Ligation products were transformed into E. cloni®10G Elite competent cells (Lucigen) by electroporation. Overnight grown colonies were picked and arrayed in 384-well format into liquid LB medium supplemented with kanamycin using a colony picking robot (Molecular Devices QPix 420). After incubation overnight at 37°C, plates were replicated in duplicate onto LB agar plates supplemented with kanamycin. The first plate served for the initial drug assay described below to identify plates that contain drug-metabolizing gain-of-function hits. The second plate was stored at 4°C for use in the secondary assay to localize drug metabolizing clones within active plates as described below. Primer #3–4 were used for Sanger sequencing of representative library clones and identified drug-metabolizing clones.
3
High-throughput Yeast Clone Sequencing
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Sorted yeast populations were plated on SD -Leu and 8–32 colonies per plate were picked into 2 mL microplates either by hand or using a QPIX 420 (Molecular Devices) automatic colony picker. Cultures were grown at 1000 rpm at 3 mm orbit at 30°C overnight. Cells (20 μL) were transferred to a fresh microplate and washed with 1 mL TE buffer (10 mM Tris, 1 mM EDTA). Cells were incubated with 20 μL zymolyase solution (5 mg/mL zymolyase, 100T in TE) at 37°C for 1 hour. Cells (5 μL) were then used in colony PCR to amplify the paired heavy and light chains. Amplicons were column purified with the Wizard SV 96 PCR Clean-Up System (Promega, cat. no. A9342) and yields were quantified with a Nanodrop spectrophotometer or the Quant-it Broad-Range dsDNA kit (Invitrogen, cat. no. Q33130). Approximately 10 ng (2.5–5 μL) of purified PCR products were then subjected to Sanger sequencing.
4
Bacterial scFv Expression and Purification
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Single colonies of E. coli TG1 cells (Lucigen, cat. 60,502) containing phagemid vectors expressing scFvs under control of a Lac promotor were picked by robot (Qpix420, Molecular Devices) into 96-well PP U-form microplates (Greiner, cat. 650,201) containing 200 µL of Terrific Broth (TB), 2% glucose, 100 μg/ml ampicillin and cultures were grown overnight at 30 °C with shaking at 750 rpm (70% humidity). Turbid overnight cultures were used to inoculate 170 μl TB containing 0.1% glucose and 100 μg/ml ampicillin using QRep 96 Pin Replicators (Molecular Devices, cat. X5051). The cultures were grown in 96-well PP U-form microplates with shaking at 30 °C for 6 h until the OD600 reached 0.6. The expression of scFvs was induced by adding 50 μl per well of 440 µM Isopropyl-β-D-thiogalactopyranoside (IPTG; Merck, cat. 420,322) to achieve a final concentration of ~ 100 μM. Expression was allowed to continue for 16–18 h at 30 °C with shaking at 750 rpm, 70% humidity before centrifuging plates to allow the harvesting of clarified media for subsequent binding assay.
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