Biological Program for Selected Senior High School
Students in Academia Sinica
!"#$%&!'()*+,'-./012#$34
Cloning of A Novel Fungal
Cellulase Candidate Gene.
Authors
Hsuan-An Chen 567 (96019)
Hung-Wei Liu 89: (96104)
Taipei Municipal Jianguo High School ;<!*, 2010
Primary Investigator: Dr. Wen-Hsiung Li =>?
Biodiversity Research Center & Genomics Research Center,
Academia Sinica
February, 2010
Cloning of A Novel Fungal
Cellulase Candidate Gene.
Hsuan-An Chen !"# & Hung-Wei Liu $%&
Taipei Municipal Jianguo High School '()*, 2010
FINAL REPORT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE BIOLOGICAL PROGRAMS FOR SELECTED SENIOR HIGH SCHOOL STUDENTS IN
ACADEMIA SINICA
Primary Investigator: Dr. Wen-Hsiung Li +,Biodiversity Research Center & Genomics Research Center,
Academia Sinica
February, 2010
Abstract
Knowing that the fossil fuel resources are getting fewer and fewer, scientists want to
develop new alternative resources. Biofuel is one of the choices. In this project we had
cloned a fungal cellulase candidate gene, the W5-CAT13 which was predicted from the
whole-genome sequence of W5, a new endemic fungus species in Taiwan. The goal of the
Li Lab and consortium’s multi-component biofuel project is to engineer microorganisms
to produce cellulosic biofuel for energy purposes. Cellulases are therefore the key
machinery that digests agricultural wastes to transform cellulose into glucose which
then feeds fermentative microorganisms to produce alcohols. We performed several
methods to amplify, clone, screen and select W5-CAT13 clones, including polymerase
chain reaction (PCR), TA cloning, blue/white selection, colony PCR, DNA sequencing
and sequence analysis. The purpose of these procedures was to assure correct sequence
of the clone to be expressed and assayed. As a result, however, we found that the gene
fragments in our experiment was contaminated, possibly from some materials
commonly used in our lab.
Introduction
To ease fossil-fuel dependency and to take advantage of the solar energy stored in
plants , especially agricultural wastes -- the part that is inedible and usually thrown
away, a major research project in the Li Wen-Hsiung Lab is in the development of
cellulosic biofuels. The goal is to transform those previously abandoned agriculture
wastes, such as rice straw or bagasse into biofuels, including bio-ethanol or other higher
alcohols, via microbial cellulose hydrolysis and fermentation activities. This approach
will also help to reduce pollution and the amount of carbon dioxide released into the
atmosphere, since the incineration of these agricultural wastes is avoided. Figure 1 (US
Department of Energy, 2009) illustrates the concept of carbon and energy cycle in the
environment with an emphasis in biofuel.
The basics of cellulase for bio-fuels
Cellulase is roughly categorized into three classes (Figure 2) (Wikipedia, 2009),
including (1) endocellulase that cuts cellulose from 3D to 2D structure, (2) exocellulase,
which cuts those long chains into double sugars, and (3) cellobiase (beta-glucosidase)
turing cellobiose or cellutetrose into glucose. The idea is to engineer these three
cellulases for efficient hydrolysis of the rich cellulose in rice straw, bagasse, etc. to
glucose, which can then serve as carbon source for natively fermentative
microorganisms, e.g., the yeast, for the production of bio-alcohols for energy purposes.
The issue is that there are presently no proper cellulases for such application.
Insufficient enzyme efficiency of currently available cellulases leading to significant
pretreatment, costs and energy required for industry-scale production renders cellulosic
biofuel impracticable based on current technology.
1
Figure 1. Carbon dioxide and biofuels in the energy cycle. (US Department of Energy, 2009)
2
Figure 2. Three major classes of cellulase and their substrates. (Wikipedia, 2009)
3
The quest for “ideal” cellulases
In order to develop new, applicable cellulases for large-scale energy production, the
approach taken in the Li Lab and consortium was to first identify new cellulase
candidates from naturally competent organisms. Through the collaboration with Prof.
Yo-Chia Chen at National Pingtung University of Science and Technology, we obtained a
new endemic fungus species in Taiwan, the W5, isolated from the rumen of Taiwan
yellow cattle. A whole-genome de novo sequencing was performed on W5; cellulase gene
candidates in W5 genome were predicted based on available cellulase gene sequences
and enzyme structures. Primers against these cellulase gene candidates were designed
for PCR amplification. Each candidate was then amplified from W5 total cDNA, TAcloned for sequence verification, and cloned into an expression vector for functional
characterization and enzyme assay in the yeast system (Figure 3).
Among all the predicted genes, the one we worked on for our research project was W5CAT13, a candidate beta-glucosidase gene. We used CAT13 gene-specific primers to PCR
amplify the gene fragments, initially TA cloned the PCR products to the commercially
available pGEM®-T Easy Vector to facilitate sequencing verification, and subsequently
the clone with correct sequence will be cloned to a yeast expression vector using the
method by Shih et al. (Shih et al., 2002) for future enzyme studies in the lab (Figure 4).
Materials and Methods
This section contains the detail information for the experimental procedures performed
during our training session in the laboratory for the purpose of W5 fungal cellulase
candidate gene cloning.
4
Figure 3. The flow chart illustrating the overall procedure for the development
of novel cellulase for the purpose of biofuel production.
5
Gene-specific PCR primer
design based on the
results of whole-genome
sequencing & gene
prediction
PCR amplify the gene of
selected candidate
cellulase
Clone the purified gene
fragments into an
appropriate vector for
sequencing verification
(Gel) purify PCR
products
Express the candidate
cellulase in selected host for
activity assay and biochemical
analysis
Figure 4. The flow chart highlighting the steps implemented in this research for
molecular cloning of a novel cellulase candidate, W5-CAT13.
6
Gene-specific primers, Polymerase chain reaction and DNA sample
preparation
Polymerase chain reaction (PCR) was first carried out using two pairs of primers (CAT13
1-1 forward and reverse, and CAT13 1-2 forward and reverse) specific to the candidate
beta-glucosidase gene, W5-CAT13 (gene specific regions: ATG AAG ACT CTT ACT TTA
TTT AC for the forward primer and GTT TTG TTC AAC ATT TTC AAG G for the
reverse). Both pairs share the same gene-specific regions, whereas the 1-1 forward
primer contains the Watson strand of EcoRI restriction site on the 5’-end, and the 1-2
reverse primer contains the Crick strand of XhoI restriction site also on the 5’-end (Shih
et al., 2002). Additionally, the 1-1 reverse primer carries a guanosine (G) on the 5’-end
and the 1-2 forward primer carries a cytidine (C) also on the 5’-end, to compliment each
of the EcoRI and XhoI restriction sites. These restriction sites were designed to facilitate
molecular cloning strategy designed and described by Vice President Andrew H.-J.
Wang’s Lab (Shih et al., 2002).
The previously synthesized W5 cDNA library prepared from W5 mRNA served as the
template DNA in this experiment. Although no rigorous annealing temperature profiling
was performed, the annealing temperature for both 1-1 and 1-2 pairs of CAT13 primers
were set at 35°C for this experiment, after a few initial attempts. PCR products were
resolved in 0.8-1% TAE agarose gel after electrophoresis, and visualized under UV light
by either ethidium bromide or SYBR® Safe (Invitrogen, USA) staining. DNA fragments
appeared at the expected size (2000 bp) relative to the DNA markers were considered
positive PCR results.
Prior to subsequent TA cloning or sequencing procedure, PCR product purification was
performed using the commercially available QIAquick spin method by QIAGEN (USA).
7
When considerable amount of non-specific PCR products were observed, gel extraction
of the 3-kb DNA fragments was done also using the QIAquick spin method. The
spectrophotometry was used to determine the OD260, OD280 and OD230 values of the
purified samples in order to estimate the quantity and quality of the products.
TA cloning and clone screening
Purified PCR products were cloned to pGEM®-T Easy Vector via a commercially
available TA cloning method (Promega, USA) and transformed to HIT-DH5-alpha E.
coli competent cells (Real Biotech Corp., USA) for subsequent sequencing analysis.
Positive transformants carrying the pGEM-T Easy Vector were Ampicillin resistant; the
clones with the inserted DNA fragment should grow into white colonies on solid media
containing X-gal (with IPTG).
Colony PCR was performed on the white colonies to screen for clones containing W5CAT13 insert. Plasmid miniprep (QIAGEN, USA) was performed on the clones to
harvest the vectors potentially with insert. The spectrophotometry was applied to
estimate the quantity and quality of the plasmid samples. The plasmids were restriction
digested at 37°C for four hours by NdeI (4 unit/ug DNA) (NEB, USA) and SacII (4 unit/
ug DNA) (NEB, USA) then heat inactivated at 65°C for 20 minutes to further confirm
the insert. 6 uL of the digested products were loaded and resolved on agarose-TAE gel
after electrophoresis. Each clone was also submitted for DNA sequencing done by a local
vendor.
DNA sequence analysis
The candidates of pGEM®-T Easy Vector with W5-CAT13 insert were each sequenced
with SP6 Promoter Primer, T7 Promoter Primer, the gene-specific (1-1 or 1-2) forward
and reverse primers, as well as a forward primer internal to W5-CAT13 gene
8
(CAT13_601f) respectively. All the sequencing results were first visually examined on
their chromatograms for an overall quality control. The sequence reads were aligned,
using ClustalW multiple sequence alignment algorithm (Larkin et al., 2007), against the
reference W5-CAT13 sequence previously obtained in W5 whole-genome sequencing.
BLAST (Altschul et al., 1990) was used to identify the regions among the reads that were
not well aligned with the reference sequence (or the vector backbone) by an NCBI DNA
sequence database search.
Results and Discussion
This study is part of the biofuel research project carried out in the Wen-Hsiung Li Lab,
the aim of which is to identify and characterize novel cellulases from W5, an endemic
fungal species in Taiwan. The ultimate goal is to apply these newly found enzymes in
cellulosic biofuel production, via the hydrolysis of agricultural wastes, such as rice straw,
and fermentation, using yeast as the host organism. We attempted to clone a predicted
beta-glucosidase gene, W5-CAT13 using the approach published by Vice President
Andrew H.-J. Wang and colleagues (Shih et al., 2002). We were able to perform the
procedures from PCR amplification of the gene, TA cloning, through DNA sequencing
for clone verification. In this section, we detail the major results of our experiments and
elaborate what we have observed.
PCR and nucleic acid purification
Although there was no rigorous testing of PCR temperature profiles, the annealing
temperature of 35°C for both 1-1 and 1-2 primer pairs was applied after several attempts.
Both 1-1 and 1-2 PCR products at the size of 2 kb were observed (Figure 5). The initial
quantity, nevertheless, was low, as only a faint band appeared on the agarose gel. We
9
3kb
2kb
Figure 5. An ethidium bromide-stained 1% agarose-TAE gel showing
the presence of PCR products at the expected size of about 2kb.
10
thus performed several sub-PCRs using products from previous reactions as the
template to obtain sufficient amount of DNA for subsequent cloning. Since the
annealing temperature is significantly lower than the primers’ predicted melting
temperatures, non-specific amplification is likely to occur. Should non-specific DNA
fragments be produced, they would be further enriched in the subsequent sub-PCR.
Cloning of the PCR-amplified gene fragments into individual sequencing vector followed
by sequencing verification is therefore highly recommended prior to molecular cloning
for gene expression and enzyme assays.
Purification of the PCR products for TA cloning were done using a commercially
available method (QIAGEN, USA). The concentrations of most of the products were
about 300 ng/uL, determined by spectrophotometry, usually in the volume of 30 uL.
TA cloning and clone screening
Only a few white colonies appeared after the W5-CAT13 1-1 TA cloning and
transformation reaction. Even fewer white colonies were obtained for sample 1-2. Most
of these clones were actually blue ones after patching (Figure 6). Such low cloning and
transformation efficiency was likely to be the result of bad DNA quality. Although the
apparent concentration appeared sufficient at ~300 ng/uL, the actual products might
include non-specifics such as primer dimers, primer extensions, etc., as a bright band or
bright smears at lower molecular weight were constantly observed. The final
concentration of true working 1-1 or 1-2 W5-CAT13 DNA molecules could have been
overly diluted for TA cloning.
Colony PCR with the same W5-CAT13 1-1 or 1-2 primer pairs was used in the initial
screening of the TA clones. Surprisingly, the colony PCR products of all screened clones
11
Figure 6. Single-colony patches of W5-CAT13 candidate TA clones.
Blue indicates the absence of an insert. White patches are clones
potentially carrying an insert.
12
were in the size of 3 kb, rather than the original 2 kb, after several attempts and at least
one repeated cloning reaction. To further verify (the presence of) the insert, we obtained
the plasmids of each of the TA clones using a commercially available miniprep method
(QIAGEN, USA) and subjected these plasmid samples to both DNA sequencing and
restriction enzyme digestion. The part on sequencing will be discussed in the next
section. The banding patterns of the digested TA clones are shown in Figure 7. We used
NdeI and SacII, each of which respectively has one restriction site on position 97 bp and
49 bp on the multiple cloning site flanking the insert on pGEM®-T Easy Vecvor (3051
bp, Figure 8) but does not cut the W5-CAT13 insert, our candidate gene of interest. We
performed one double-digestion (NdeI and SacII, lane 1), two single-digestions (NdeI or
SacII alone, lanes 2 and 3), as well as a no-enzyme (water) control (lane 4) (A 2-log DNA
ladder was used and loaded on the left.) As seen on the gel, both single- and doubledigestions resulted in a single major band at ~3 kb, whereas two bands, one at ~2.5 kb
(between 2 and 3 kb of the 2-log ladder) and the other at ~5 kb, were observed in the noenzyme control. The 3-kb fragments in the singly-digested samples were likely to be the
linearized plasmid; those in the doubly-digested reaction should also be the linearized
plasmid, with or without the insert if the insert had been 3 kb. We were more inclined to
conclude that this clone did not contain any inserts that are larger than 0.5 kb in size, as
(1) no significant increase in the brightness (DNA quantity) of the 3-kb band as should
be if the insert were 3 kb, or (2) no appearance of bands in any size other than 3 kb was
observed in lane 1 (double digestion) where the insert should be cut off from the vector
backbone, relative to lanes 2 and 3 (single digestion) thus indicating the absence of DNA
fragments larger than 500 bp.
13
6kb
1 2 3 4
3kb
1kb
Figure 7. Restriction digestion profile of one CAT13 pGEM-T Easy
clone (1. NdeI & SacII, 2. NdeI, 3. SacII, 4. water).
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II.C. pGEM®-T Easy Vector Map and Sequence Reference Points
XmnI 2009
f1 ori
Ampr
pGEM-T Easy
Vector
lacZ
T
(3015bp)
T
T7
ApaI
AatII
SphI
BstZI
NcoI
BstZI
NotI
SacII
EcoRI
SpeI
EcoRI
NotI
BstZI
PstI
SalI
NdeI
SacI
BstXI
NsiI
➞
ori
SP6
1 start
14
20
26
31
37
43
43
49
52
64
70
77
77
88
90
97
109
118
127
141
1473VA05_6A
NaeI 2707
➞
ScaI 1890
Figure 3. pGEM®-T Easy Vector circle map and sequence reference points.
Figure 8.® The map and sequence reference points of pGEM®-T Easy
pGEM -T Easy Vector sequence reference points:
Vector used for TA cloning in this experiment (Promega).
T7 RNA polymerase transcription initiation site
multiple cloning region
SP6 RNA polymerase promoter (–17 to +3)
SP6 RNA polymerase transcription initiation site
pUC/M13 Reverse Sequencing Primer binding site
lacZ start codon
lac operator
β-lactamase coding region
phage f1 region
lac operon sequences
pUC/M13 Forward Sequencing Primer binding site
T7 RNA polymerase promoter (–17 to +3)
1
10–128
139–158
141
176–197
180
200–216
1337–2197
2380–2835
2836–2996, 166–395
2949–2972
2999–3
Note: Inserts can be sequenced using the SP6 Promoter Primer (Cat.# Q5011), T7
Promoter Primer (Cat.# Q5021), pUC/M13 Forward Primer (Cat.# Q5601), or
pUC/M13 Reverse Primer (Cat.# Q5421).
!
Note: A single digest with BstZI (Cat.# R6881), EcoRI (Cat.# R6011) or NotI (Cat.#
R6431) will release inserts cloned into the pGEM®-T Easy Vector. Double digests can
also be used to release inserts.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM042
Page 6
Printed in USA.
Revised 5/07
15
DNA sequence analysis
We used five primers to sequence the TA clone of interest. Two of the primers were the
vector-based T7 and SP6 Promoter primers flanking the multiple cloning site (Figure 8).
Since the W5-CAT13 gene is expected to be 2 kb in size, an internal gene-specific
forward primer at gene position 601 bp was also used, in addition to the forward and
reverse PCR primers. The reverse complimentary sequences of the sequencing data
generated with a reverse primer were used in sequence alignment and analysis.
Although vector-based T7 and SP6 Promoter primers generated quality sequencing
results, the gene-specific primers gave bad or no reads. The 5’ part of the sequence reads
from T7 and SP6 Promoter primers aligned well with pGEM®-T Easy Vector flanking
the cloning site, indicating the vector backbone was intact and the sample was free of
contamination. None of the sequence data, however, could align with W5-CAT13,
suggesting no successful insertion of this candidate gene.
In order to identify the DNA fragment being inserted and sequenced on pGEM®-T Easy
Vector, we did a BLAST search (Altschul et al., 1990) against the entire available NCBI
DNA database (nr). Figure 9 is an excerpt showing the top hits from the search result. In
particular our submitted query sequence (190 bp) was 100% identical to 81% of a partial
16S rRNA gene from an uncultured bacterium (accession number FM242723.1) with the
Expect value (E) of 5e-40, suggesting the short insert carried in this particular clone
examined was likely to be the result of a random amplification. Other top hits include
uncultured fungi partial 18S rRNA gene, and some cloning vector backbones. Although
most of these BLAST hits were short thus we were unable to determine the source of this
16
Figure 9. An excerpt of BLAST result, showing top hits with significant alignments.
17
false insert, the BLAST results suggested that this inserted fragment was likely to be
from an arbitrary lab contaminant.
Summary
In this experiment, we have learned not only the basic molecular biology techniques and
skills in cloning and manipulation of genes, but also some advanced knowledge in
modern biology and bio-fuel. We have also learned to adjust our expectations and goals
on our research, as most of great scientific findings are constituted by many small parts,
which is the role of our project in the Li Lab. As much as we were eager to finish this
experiment by ourselves, we have come to realize that research is a challenging
endeavor -- it not only takes hard work, but also requires considerable patience and
luck. Making every effort to succeed on our experiments, we spent a lot of time working
in the lab, trial and error, and repeating many steps whose significance did not seem
obvious at that moment. There were times when our ambition was running down; the
idea of giving up surfaced on our minds. Eventually, however, we overcame the
obstacles and tiredness and found the true enthusiasm for doing research. Regardless of
the outcome of our experiments, we still enjoyed the science through the execution of
the research project. This experience may be trivial to our colleagues in the lab, but it is
significant to us, who step into this territory for the very first time. It has been a
profound experience in our senior high school life. Thank you to those who have helped
us and taught us the attitude of mind for doing scientific research.
Acknowledgements
We would like to thank Dr. Christine Wang for teaching us how to conduct the
experiments, offering us invaluable advice and helping us with the editing of this report.
18
We are also grateful to Dr. Tzi-Yuan Wang !"# and other colleagues in the Li lab, for
giving us support and advice. We would like to express our sincerest gratitude to Dr.
Wen-Hsiung Li $%& for having us in his lab where we could carry out experiments for
our training and research program. It has been a great pleasure to be part of the
Biological Programs for Selected Senior High School Students in Academia Sinica. We
would like to thank all the faculty members, program officers, and participant institutes
and departments for this wonderful training program.
References
Altschul et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215: 403-10.
Larkin et al. (2007) ClustalW and ClustalX version 2. Bioinformatics 23: 2947-2948.
Shih et al. (2002) High-throughput screening of soluble recombinant proteins. Protein
Sci. 11: 1714-1719.
US Department of Energy (2009) http://www.jgi.doe.gov/education/bioenergy/
bioenergy_1.html.
Wikipedia (2009) http://en.wikipedia.org/wiki/Cellulase.
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