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.
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.
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
2. Measuring the Growth of E. coli Clones
3. Addition of the Assay Mix to Each Plate
4. Reading Absorbance of Assayed Clones
5. Representative Results
An example of absorbance readings from a single 384 well plate containing a positive clone is shown in Figure 2. Positive clones show a marked increase in absorbance over those not expressing cellulase activity. Differences in assay time, well location on the plate, or DNP concentration (may be introduced by filtering out un-dissolved DNP) can affect absolute absorbance readings. Relative absorbance readings, such as the difference in absorbance above the plate average or column average, are a more robust method of identifying cellulase positive clones.
Following identification of positive clones from the library plates, it is recommended to replicate all positive clones into a new plate for secondary screening. This eliminates effects arising from well location or plate variation and allows for more direct comparison between positive clones.
Figure 1. Flow chart of the high-throughput assay for a metagenomic library cloned into E. coli and stored at -80°C.
Figure 2. Absorbance readings from one 384-well plate containing a positive clone. Cellulase positive clones can be identified by significantly increased absorbance over negative clones.
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 unsupervised screening of more than 25 384-well plates per hour. The data can be easily exported from the software into a Microsoft Excel spreadsheet, allowing for analysis or processing by third party software.
One limitation of this assay exists in the expression potential of exogenous proteins in the E. coli host. A metagenomic library contains DNA from many different organisms, only a subset of which can be recognized by the E. coli transcription/translation machinery. Even when expressed, exogenous gene products may not achieve functionality due to improper folding, processing, or inadequate expression levels. These limitations can be partially offset through the utilization of copy control systems, as seen in previous functional metagenomic screening studies. 12. The pCC1 copy control fosmid used here allows induction through addition of the inducer L-arabinose, and increases copy number from one, to up to 100 copies per cell 13. These systems can improve the outcome of activity-based screens by enabling single copy growth for stability with subsequent induction for increased activity.
The substrate 2,4-DNP-Cellobioside used in our screen is not commercially available from suppliers, but similar substrates can be purchased. Sigma-Aldrich offers 2-nitrophenyl (Cat No. N4764) and 4-nitrophenyl (Cat No. N5759) cellobiosides. The general screen as described could be undertaken with these substrates, but some modifications would be required. These mono-substituted phenols have higher pKa values, around 7.2, compared to DNP, which is around 4. Optimum pH for cellulase activity has been reported to range from pH 4.5-6.0 14, 15. The use of DNP-C allows for the assay to be carried out at optimal pH conditions, allowing for easier identification of cellulases. In addition, the di-substituted glycoside is much more reactive than the others, allowing for the detection of more reluctant cellulases. Thus, the use of DNP-cellobioside has allowed for a more robust and sensitive screen than would be available with commercial substrates.
It is notable that this screen can be used for detection of any enzymes with an associated colorimetric or fluorometric substrate. Cellulases are a stable and active enzyme, ideal for the initial development and optimization of the screening parameters. The general approach presented here is a powerful tool for the screening of metagenomic libraries for both academic and industrial applications.
The authors have nothing to disclose.
The authors would like to thank Dr. Steve Withers and Hong-Ming Chen for providing DNP-Cellobioside substrate.
Material Name | Typ | 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 |