A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Summary

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Abstract

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production ¹. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils ², buffalo rumen ³ and the termite hind-gut ⁴ using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood ⁵). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio ⁶. Other methods have been reported ^7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate ⁹. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction ¹⁰. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

Protocol

Before starting this protocol, you will need your metagenomic library stored in a 384 well plate format. In our study, we used the pCC1 copy control fosmid vector in combination with phage T1-resistant TransforMax EPI300-T1R E. coli cells as the library host and stored our plates at -80°C 11. 1. Replication of the Metagenomic Library Plates Defrost the plates containing your library at 37°C for approximately 20 minutes, or until all wells …

Discussion

A high throughput screen for the rapid detection of cellulolytic activity from a large insert genomic DNA metagenomic library expressed in E. coli is described in this protocol. This method is an improvement over the CMC/Congo Red assay commonly used in the literature. It is solution based, and allows for one-pot chemistry screening in 384-well plates, with the final output as absorbance readings from a plate reader allowing for quantitative analysis. The automation of each step of this process allows for the un…

Materials

Material Name	Type	Company	Catalogue Number	Comment
qPix2		Genetix		With 384-pin gridding head
qFill3		Genetix		With 384-well manifold
Varioskan		Thermo-Fisher
RapidStak		Thermo-Fisher		Connected to Varioskan
Micro90 Detergent		Cole-Parmer	18100-00	Diluted to 2% in water
Ethanol		Major Lab Supplier		Diluted to 80% in water
Chloramphenicol		Sigma	C0378	12.5mg/mL in ethanol
LB broth, Miller		Fisher	BP1426-2	25g/L, autoclaved
384-well flat bottom plates		Corning	3680
L-(+)-Arabinose		Sigma	A3256	100mg/mL in water
Potassium Acetate		Fisher	P171	50mM in water, autoclaved, adjusted to pH 5.5 with HCl
Triton X-100		Fisher	BP151
Trizma hydrochloride		Sigma	T3253	In TE buffer solution, 100mM
EDTA disodium salt		Sigma	E5134	In TE buffer solution, 10mM
2,4-dinitrophenyl cellobioside				Provided by Dr. Steve Withers, UBC
Dimethyl Sulfoxide		Sigma	D8418