Transcript Cloning (library construction)

Environmental DNA Libraries
METAGENOME
Ju Hyoung Lim
Nov 24, 2003
Definition of Metagenome
Meta-. Gk. meta, of change, beyond.
[Handelsman et al. (1998) Chem Biol 5: R245-R249]
• The genomes of the total microbiota found in nature
• Environmental DNA comprising the genetic blueprints
of entire microbial consoritia
• The collective genomes directly cloned from all
microorganisms present in a habitat at a certain time
point
• DNA libraries representing the genome of uncultured
microbes, as a rich source for isolation of many novel
genes
Historical background
Pure cultured biota VS Total microbiota
• The great plate count anomaly (Staley et al. 1985)
• Loss of realistic prokaryotic biodiversity in culture
method
• Limitation of finding novel genes and protein when
using culture method
Historical background
1. The great plate count anomaly (Staley et al. 1985)
1g of Soil
plate
cultivation
4,000 species
40 species
• 1g of soil contains up to 4,000 different spp. but less than 1% are
readily culturable w/ known cultivation method.
• The culturability, i.e. the ability to grow as colonies on a rich
culture medium is extremely low for natural populations of bacteria.
• The reasons remain obscure but it is not difficult to understand that
many organisms are not capable of adapting to the artificial and
rather restrictive conditions of laboratory pure cultures.
Historical background
1. The great plate count anomaly (Staley et al. 1985)
Total vs. cultivatable microbial diversity. Left, microbes on a soil flake
were stained with DAPI (4’,6-Diamidino-2-phenylindole) and detected
using fluorescence microscopy. Right, colonies grown on enriched LB
agar. There seems to be difference between two cases at 2-3 orders of
magnitude. (from Lorenz and Schleper, 2002, J Mol Catal, B Enzym
19-20:13)
Historical background
2. Loss of realistic prokaryotic biodiversity in culture method
• Traditional cultivation method may result in the loss of major
portions of the microbial communities
• Even closesly related bacterial species need very different culture
conditions.
97.7%
• Phylogenetic study with 16S rDNA amplification from
environmental DNA (by Pace and colleagues in 1986) : revealed an
astonishing number of new microbial groups that had never been
picked up by cultivation.
Historical background
3. Limitation of finding novel genes and protein when using
culture method
Soil
plate
cultivation
1,000 novel
genes
10 novel genes
<Hitherto identified novel enzymes by
using metagenomic studies (2002)>
Historical background
• Torsvik and Goksoyr (1978)
1990’s
to illustrate
novel microbial diversity
- Environmental DNA
• Pace et al. (1986)
- 16S rDNA from soil DNA
• Schmidt et al. (1991)
- 16S rDNA from aquatic DNA
• Handelsman et al. (1998)
- Metagenomic library
TREND
Overview of metagenome construction
• Direct extraction of genomic DNA
from environmental samples (marine
and soil)
• Choice of an appropriate vector
• Cloning (library construction)
• Screening and further analysis
<A Schematic Comparison of the
cultivation
and
metagenomic
approaches
to
obtain
novel
biocatalysts (from Lorenz et al.,
2002)>
Metagenomic library construction
• Isolation of environmental DNA
• Choice of an appropriate vector
• Cloning (library construction)
• Screening and further analysis
Isolation of environmental DNA (soil)
• Marine environment is easier to access than soil environment.
• Up to 40 Kb or even more than 100 Kb DNA fragments have been cloned and
they have served to characterize novel genomic fragments of abundant marine
archaea and bacteria (the late 1990’s).
• Soil environments is more difficult to prepare metagenomic libraries because
of the presence of high MW inhibitor such as humic acid, fulvic acid,
polyphenolic compounds in soil samples -> poor quality and quantity of DNA.
Therefore numerous strategies were developed.
Soil DNA extraction: basically falling into two ways…
lysis
Mechanical
in situ extraction
agitation
lysis w/o
mechanical
agitation
Physical
separation
ex situ extraction
Isolation of environmental DNA (soil)
in situ extraction
advantage
disadvantage
purpose
ex situ extraction
• High DNA yields
• Low extraction bias
• Very large size DNA
fragment (20kb up to >500kb)
• Less comtamination of nonmicrobial and free soil DNA
• DNA is sheared to small
sizes (1kb up to 50kb).
• More Contamination
• Low DNA yields
• High extraction bias
• Cloning into plasmid or
• Cloning into BAC or
lambda vector for one gene to cosmid vector for large geneshort gene-cluster expression cluster expression test.
test.
Isolation of environmental DNA (soil)
<Different DNA isolation procedures produce
different fragment sizes. Left, in situ extracted,
smaller, heterogeneous in size. Right, ex situ
extracted. DNA was PFGEed and EtBr-stained.
(from Lorenz and Schleper, 2002)>
Metagenomic library construction
• Isolation of environmental DNA
• Choice of an appropriate vector
• Cloning (library construction)
• Screening and further analysis
Purpose VS choice of vector
Short size insert
(<10kb)
Purpose
Sequencebased
approach
Activity-based
approach
Normal
plasmid or
lambda vector
Plasmid
possessing
artificial
promoter
Vector
• pBluescript
• General T
vector
• pT7 vector
• plac vector
Long size insert
(10kb up to 100kb)
Sequencebased
approach
Activity-based
approach
Long-insert maintenance vector
• BAC
• Fosmid
• Cosmid
Sequence-based approach
• PCR-amplification for searching specific genes using conserved primer
• Southern hybridization using conserved-sequence probe
clone 2148
clone 25
- clones
clone 124
+ clones
- It is rather conservative method, so the range of discovery could be
narrow. Yet molecular diversity is so great that numerous novel enzymes
can be retrieved in this way.
- Long-insert cloning is better for this method
- Vector: BAC, cosmid, fosmid,…>>…, plasmid, lambda vector
Sequence-based approach (continued)
• Partial or full sequencing -> ORF assigning and blast
<Physical map of a cosmid clone identified in oral metagenome (from
Voget et al. 2003)>
- useful to identify an entire gene clusters encoding mulifunctional modular
enzyme e.g. polyketide synthases occur in clusters exceeding 100 kbp of
contiguous DNA.
- Vector: BAC, cosmid, fosmid,…>>…, plasmid, lambda vector
• Advantage of sequence-based approach:
- It overcomes possibilities of excluding some enzymes which are
expressed at subthreshold level or not expressed in the heterogenous host.
- Incomplete genes resulted from partial cloning can be detected.
Activity-based approach
• Artificial facilitation of gene expression
ribosome
P
transcript
Cloning
Transformation
into
Specific
expression
substrate
of surrogate
vector possessing
strong
expression
promoter
containing
host
for artificial
E.
medium
coli transcription
- Useful for identifying single gene (1-2 kb) to small gene cluster
- It overcomes low expressibility in surrogate host
- Only short inserts are possible with this method.
- Vector: Plasmid expression vector such as pT series vector
Activity-based approach (continued)
• Gene expression from insert-borne promoters
clone 25
Specific substrate
containing medium
- Useful for identifying single gene to large clusters
- Eugenes, not pseudogenes, can be identified.
- Problem of heterologous expression
- Vector: BAC, cosmid, fosmid
History of BAC vector
• Larger the insert DNA, more difficult maintenance of the clone
- Extra energy for replication of so long non-chromosomal DNA
- Deleterious recombination events
• More copy number of vector, easier the loss of the extrachromosome
• Normal plasmid ori can’t afford to very long stretch of replicon.
YAC (yeast artificial chromosome) vector (Burke et al. Science 1987)
• developed to maintain clones with large sizes (>500kb)
• However, fatal instability (YAC clone rescue failure) and chimera
problems have been observed.
BAC (bacteria artificial chromosome) vector (Shizuya et al. PNAS 1992)
• capable of maintaining 1,000 kb fragment. 1-2 copies.
• Insert DNA is stable, easy to manipulate, low recombination
YAC vs BAC
Features
YAC
BAC
Configuration
Linear
Circular
Host
Yeast
Bacteria
Copy Number / Cell
1
1-2
Cloning Capacity
Unlimited
up-to 350 kb
Transformation
Spheroplast (107 T/ug)
Electroporation (1010
T/ug)
Chimerism
up to 40%
None to low
DNA Isolation
Pulsed-field-gelelectrophoresis Gel Isolation
Standard Plasmid
Miniprep
Insert Stability
Unstable
Stable
Available BAC vectors
• based on E. coli F factor, which strictly control its replication and copy
number (1-2)
Available BAC vectors (continued)
Name
Cloning sites
Recombinant
selection
pBAC108L
(6.7 kb)
HindIII, BamHI
no
Shizuya et al., 1992
pBeloBAC1
1
(7.4 kb)
HindIII,
BamHI, SphI
lacZ
Kim et al., 1996
pECSBAC4
(9.3 kb)
EcoRI, HindIII,
BamHI
lacZ
Frijters et al., 1996
BIBAC2
(23.5 kb)
BamHI
sacBII
Plant Transformation
via Agrobacterium
Hamilton et al., 1996
pBACwich
(11 kb)
HindIII,
BamHI, SphI
lacZ
Plant Transformation
via Site-Specific
Recombination
Choi et al.,
unpublished
pBACe3.6
(11.5 kb)
BamHI, SacI,
SacII, MluI,
EcoRI, AvaIII
sacBII
High copy number is
available
de Jong et al.,
unpublished
pClasper
(9.7 kb)
homologous
recombination
in yeast
LEU2
Yeast and bacteria
shuttle vector
Bradshaw et al., 1995
Features
Reference
Metagenomic library construction
• Isolation of environmental DNA
• Choice of an appropriate vector
• Cloning (library construction)
• Screening and further analysis
Cloning (library construction)
• Insert preparation
1. Size fractionation
- Pulse field gel electrophoresis (PFGE) & gel extraction of appropriatesized fragements
- Restriction enzyme cutting or mechanical shearing
2. Polishing ends
- Blunt ends formation (polymerase filling or nuclease trimming)
- Sticky ends formation (restriction digest)
- 3’ ends A-overhang (blud ends formation followed by Taq pol treatment)
• Vector preparation
- Restriction digest and 5’ end dephosphorylation
Cloning (library construction), continued
• Ligation
• Transformation
- electroporation
• Confirming insert clone, not self-ligate
• Library grouping
- Not1 digestion (5’-GCGGCCGC-3’): high G+C group/ low G+C group
- 16S rDNA amplifying and sequencing, restriction pattern, PCR fingerprint
: metagenomic diversity
• Transfer of the clones to 96-well plates and conservation
Metagenomic library construction
• Isolation of environmental DNA
• Choice of an appropriate vector
• Cloning (library construction)
• Screening and further analysis
Rondon et al. AEM 2000
• Construction of metagenomic BAC libraries from soil
- Two libraries, SL1 (mean size 27 kb) and SL2 (mean size 44.5 kb)
• Phylogenetic analysis of the libraries
- 16S rDNA sequencing
• Screening of Dnase, amylase,
producing, and antibacterial clones
lipase-
- Confirming that the clones were not duplicate
clones by using restriction pattern analysis
• Determinaton of gene loci
- transposon mutagenesis
- full sequencing
Voget et al. AEM 2003
• Laboratory enrichment of soil microbiota showing agarolytic activities
- for increasing cloning efficiency and fast isolation of specific metagenome
• Construction of cosmid DNA libraries
- Insert DNA was partially digested by Sau3A and cloned into cosmid pWE15
• Full sequencing and ORF assignment of amidase, two cellulases, αamylase, and pectate lyase genes
• PCR cloning of entire ORFs into expression vector
• Enzyme activities of protein extracts
Diaz-Torres et al. Antimicrob Agents Chemother 2003
• Oral metagenome was constructed from dental plaques and saliva
- Insert DNA was sheared by ultrasonication.
- Mung bean nuclease treated inserts were electrophoresed.
- 800 to 3,000 bp sized fragments were recovered and 3’ adenylated by Taq
pol incubation.
- Prepared inserts were cloned into TA expression vector.
• Of 450 transformants, 18 (4%) colonies were screened on LB medium
as tetracyclin resistant clones
• Confirming that if the resistance is derived from previous known
mechanism. PCR using tet gene primers.
Perspectives in metagenomic study
• The microbial world seems to offer the greatest natural resource of
molecular diversity. Classical cultivation method is valid and powerful
but severely restricted in scope. So, it needs to be complemented by
metagenomic study.
• Yet methodological problems will set the limits of this approach:
Heterologous gene expression in surrogate host is not always
successful.
• Using a variety of different hosts e.g. Streptomyces lividans and
Bacillus sp. in addition to E. coli should significantly boost the success
rate in heterologous expression screens
INDEX
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Definition of metagenome
Historical background
Overview of metagenome construction
Isolation of environmental DNA
Choice of an appropriate vector
Cloning (library construction)
Screening and further analysis
Perspectived in metagenomic study