Table 1 Compared characteristics of the gene candidates (Halo-cellulase 1 and 2) and

the profile halophilic cellulase gene, Hu-CBH1.

After using our HMM to search against our metagenomic data, we found two

candidates that have the potential to function in ionic liquids to break down

cellulose. The first gene, Halo-cellulase 1, matched the HMM the closest. It

contains a cellulase and fibronectin type III-like domain similar to the profile

gene, Hu-CBH1. The second candidate, Halo-cellulase 2, shares homology

to archaeal genes and contains only a cellulase domain.

In this study, we ran a Hidden Markov Model (HMM) against

metagenomic libraries collected in salt flats to find novel halo-cellulase

candidates.

Methods

Results

IntroductionBiofuel, as an alternative fuel source, can be produced by the

enzymatic breakdown of cellulose through ionic liquids and cellulases.

• Substrate: Cellulose

• the most abundant organic compound

• composed of metabolically versatile glucose

• Pre-Treatment: Ionic liquids

• high salt solutions used as an alternative to heat pretreatment in

order to solubilize cellulose for efficient digestion.

• Enzyme: Halophilic Cellulases

• Salt-tolerant enzymes that hasten the digestion of cellulose

• Normal cellulases are unable to work in the ionic liquid’s high

salt environment

Our research seeks to:

• Identify candidate halophilic cellulase (“halo-cellulases”) genes from

metagenomic data libraries

• Insert two strong candidates into the amplification/overexpression

vector, pTA963 (see Fig. 4) via Gibson assembly

• Amplify the expression vectors in E. coli

• Express the novel halo-cellulase candidates in the halophilic

archaeon Haloferax volcanii

• Characterize the halo-cellulase activity with a reducing sugar assay

References and Acknowledgments1. Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive

sequence similarity searching. Nucleic Acids Research 39:W29 - W37.

2. Eddy SR. (2004) What Is a Hidden Markov Model? Nature Biotechnology

22.10: 1315-316.

3. Allers T, Barak S, Liddell S, Wardell K, Mevarech M (2010) Improved strains

and plasmid vectors for conditional overexpression of his- tagged proteins

in Haloferax volcanii. Appl Environ Microbiol 76:1759–1769.

Special Thanks To: Dr. David Bernick, Dr. Hugh Olsen, Dr. Mark Akeson, Jessica

Kost, Charles Paine, College 9, Crown College, Merrill College

Identification of Novel Halo-Cellulases for Applications in Biofuel

Shadie W. Nimri, Tyler D. Ortega, Christian P. Pettet, Arjun S. Sandhu,

Kaylee C. Walker, David L. BernickUniversity of California, Santa Cruz

Biomolecular Engineering and Bioinformatics

Fig. 1 A wooden post

from metagenomic

sample site A23 in

Fremont, CA,

showing degradation

via ionic liquids

(photo credit: Wade

Dugdale).

Fig. 2 Proposed

future system for the

production of

cellulose-based

biofuel (butanol) in an

ionic-liquid system.1

Fig. 3 Hidden Markov Model

searches utilize already

known genes to create a

profile against which novel

genomic data is statistically

compared.2

Halo-cellulase 1 and 2 will each be

cloned into a modified expression

vector for Haloferax volcanii, pTA963:

• The histidine tag allows proteins

expressed on this vector to be

purified on a cobalt column.

• The E. coli origin of replication

allows the plasmids to be amplified

in the bacterium.

For our following experiment we used PCR

assembly to assemble two gene-block

fragments for Halo-cellulase 2, represented in

lanes 5 & 6. The band lengths for our product

were measured to be about 1200 bp ,as

expected, compared to the DNA Ladder

standard in lane 1.

Fig. 4 Graphical

map of the shuttle

vector, pTA963.3

• Halo-cellulase 1 and 2 will be annealed to the pTA963 expression vector

through Gibson assembly.

• The construct will be transformed into Haloferax volcanii for protein

expression.

• The protein can be purified by binding the histidine tag on the

recombinant protein onto a Ni2+ column.

• The protein will be characterized with a 3,5-dinitrosalicylic acid (DNS)

assay to detect simple sugars (cellulase product).

Future Work

Fig. 5 The assembled Halo-

Cellulase 2 gene quantified

and characterized on an

agarose gel.

Results (cont’d)

The candidates were further analyzed with several bioinformatic tools

including BLAST, Pfam, and EMBOSS tools. Through metagenomic

analysis, potential genes were filtered to result in two strong candidates

for cellulosic enzymatic activity and halotolerance.