Transcript Advanced Environmental Biotechnology II Week 14 - Gene cloning - gene

Advanced Environmental
Biotechnology II
Week 14 - Gene cloning - gene
libraries and the selection of
clones
The story so far ….
The environment is made and
maintained by living things
(organisms).
Organisms can be used to make the
environment healthier.
Organisms are chemical factories
that take materials and energy in
and transform them.
Organisms are made of cells.
Enzymes do the work of cells.
Enzymes are made of proteins, and
sometimes RNA.
Proteins and RNA are made of
smaller subunits.
Proteins are made of 20 different
amino acids arranged in order.
DNA has a code which says which
amino acids go in what order to
make an enzyme.
The DNA is made of long strings of
smaller subunits.
In many microorganisms the DNA is
kept in chromosomes.
Some DNA is also found in smaller
pieces not in the chromosome.
These smaller pieces are called
plasmids.
Plasmids can replicate.
Plasmids can move from one
microorganisms to another.
The plasmids also move their DNA,
and the codes on the DNA.
Plasmids can be used to carry DNA
codes into microorganisms.
These plasmids transform the
microorganisms.
The application of genomics and derivative technologies yields insight into
ecosystems. The use of genomics, functional genomics, proteomic and
systems modeling approaches allows for the analysis of community
population structure, functional capabilities and dynamics. The process
typically begins with sequencing of DNA extracted from an environmental
sample, either after cloning the DNA into a library or by affixing to beads
and direct sequencing. After the sequence is assembled, the computational
identification of marker genes allows for the identification and phylogenetic
classification of the members of the community and enables the design of
probes for subsequent population structure experiments. The assignment of
sequence fragments into groups that correspond to a single type of
organism (a process called ‘binning’) is facilitated by identification of marker
genes within the fragments, as well as by other characteristics such as G+C
content bias and codon usage preferences. Computational genome
annotation, consisting of the prediction of genes and assignment of function
using characterized homologs and genomic context, allows for the
description of the functional capabilities of the community. Knowledge of the
genes present also enables functional genomic and proteomic techniques,
applied to extracts of protein and RNA transcripts from the sample. These
latter studies inform systems modeling, which can be used to interpret and
predict the dynamics of the ecosystem and to guide future studies. qPCR,
quantitative polymerase chain reaction.
Molecular approaches for microbial community analysis
10
Molecular approaches for microbial community analysis
11
Today we will look at how we can
use plasmids to transform
microorganisms.
These microorganisms can then be
grown in clones.
Each clone will have a unique new
piece of DNA.
The clones can be grown to make
libraries of DNA.
Restriction enzymes
Restriction enzymes are proteins which
cut DNA.
They cut DNA whenever a specific DNA
sequence is present.
For example, the enzyme called HaeIII
cuts at GGCC.
The enzyme EcoRI cuts at GAATTC.
Different restriction enzymes cut at
different DNA sequences.
Sticky ends
Some restriction enzymes cut
across strands of the DNA
molecule to produce overhanging,
"sticky" ends.
These sticky ends are useful to join
together different DNA molecules.
Res_enz.mov
Restriction Enzymes
3. Examples of the DNA sequences that are
recognized by other restriction enzymes are
shown below.
HaeIII
5’ – G G C C – 3’
3’ – C C G G – 5’
TaqI
5’ – T C G A – 3’
3’ – A G C T – 5’
PstI
5’ – C T G C A G – 3’
3’ – G A C G T C – 5’
NotI
5’ – G C G G C C G C – 3’
3’ – C G C C G G C G – 5’
Restriction Enzymes come from
Bacteria
Restriction enzymes are used by bacteria to
protect themselves against viruses.
They restrict the growth of invading viruses by
cutting up the DNA of the virus.
Their names come from the bacteria in which they
were discovered.
EcoRI was found in Escherichia coli.
TaqI was found in Thermus aquaticus, a species
of bacterium that is found in hot springs.
DNA Ligase
DNA ligase is an enzyme that can
join (ligate) DNA molecules
together.
Restriction enzymes and DNA
ligase are used to clone DNA.
Cutting and ligating DNA
Strategies and steps in cloning.
Basic Steps -1
Cut the vector DNA with a restriction enzyme.
Cut the DNA that we want to clone with the same
restriction enzyme.
Mix together the vector DNA with the other DNA.
Add DNA ligase to ligate the DNA molecules
together.
The "sticky ends" help in joining the molecules
together with DNA ligase.
Basic Steps -2
Put these recombinant DNA molecules into
E. coli.
The vector will “transform” the bacterium to
become resistant to the antibiotic
ampicillin. This is called transformation.
Bacteria with antibiotic resistance have been
transformed with the vector and carry a
plasmid.
Basic Steps -3
Find the bacteria that carry recombinant
plasmids, i.e. plasmids that have become
combined with another DNA molecule.
This produces a collection of bacteria that
contain fragments of new DNA. This is
called a library of cloned DNA.
The basic steps in gene cloning
DNA extracted from an organism known to have
the gene of interest is cut into gene-size pieces
with restriction enzymes.
Bacterial plasmids are cut with the same restriction
enzyme.
The gene-sized DNA and cut plasmids are
combined into one test tube. Often, a plasmid
and gene-size piece of DNA will anneal together
forming a recombinant plasmid (recombinant
DNA).
Recombinant plasmids are transferred into
bacteria.
The bacteria are plated out and grow into colonies.
All the colonies on all the plates are called a
gene library.
The gene library is screened to identify the
colonies containing the genes of interest by
looking for one of three things:
the DNA sequence of the gene of interest or a very
similar gene
the protein encoded by the gene of interest
a DNA marker whose location has been mapped close
to the gene of interest
gene_cloning_in_bac.mov
plasmid_cloning.mov
http://www.whfreeman.com/lodish4e/con_index.htm?99vos
Libraries of Genes
More and more genes are being catalogued
(cloned, DNA sequence determined, and filed)
from a variety of different sources.
Many bacterial genomes have been sequenced.
A few eukaryote genomes, including human, have
also been sequenced.
It is possible to use the internet to look collections
of genes that have been cloned from several
organisms, and find the functions of those genes.
Gene Libraries - Library
Construction
A gene library can be defined as a collection
of living bacteria colonies that have been
transformed with different pieces of DNA
that is the source of the gene of interest.
If a library has a colony of bacteria for every
gene, it will consist of tens of thousands of
colonies or clones.
Screening the Library
The library must be screened to discover which
bacterial colony is making copies of which gene.
The scientist must know either the DNA sequence
of the gene, or a very similar gene, the protein
that the gene produces, or a DNA marker that
has been mapped very close to the gene.
Library screening identifies colonies, which have
particular genes.
Growing more Plasmids
When library colonies with the desired
genes are located, the bacteria can be
grown to make millions of copies of the
recombinant plasmids that contain the
genes.
Clones
Large insert clones
YACs (Yeast Artificial Chromosomes
Useful for mapping ~1mb inserts
Unstable during construction and propagation
Not useful for sequencing
BACs (Bacterial Artificial Chromosomes)
~150kb insert
Extremely stable and easy to propagate
Gold standard for sequencing targets and chromosome-scale maps
Cosmids
~50kb insert
Extremely stable and easy to propagate
Useful for sequencing but too small for chromosome maps
Sequence-ready clones
Plasmids
1-10kb insert capacity
High copy number
Easy to sequence bi-directionally
Automated clone picking/DNA isolation possible
Examples: pUC18, pBR322
Single-stranded Bacteriophage
1-5kb insert capacity
Grows at high copy as plasmid and is shed into medium as single stranded DNA
phage
Easy to isolate, pick, sequence
Easy to automate
M13 is used almost exclusively
Microbiological techniques are often
based on isolation of pure cultures
and morphological, metabolic,
biochemical and genetic assays.
They have given lots of information on
the biodiversity of microbial
communities.
We don’t know enough about the needs of
microorganisms.
We don’t know enough about the
relationships between organisms.
So we can’t get pure cultures of most
microorganisms in natural environments.
Most culture methods are good for certain
groups of microorganisms, but other
important groups do not live well.
We can use molecular biology approaches.
The techniques are based on the RNA of the
small ribosomal subunit or their genes.
Lots of this molecule are found in all living
things.
It is a highly conserved molecule but has some
highly variable regions.
We can compare organisms, and find the
differences.
The gene sequence can be easily sequenced.
In wastewater treatment, microbial
molecular ecology techniques have
been used mainly to the study of flocs
(activated sludge) and biofilms that
grow in aerobic treatment systems
(trickling filters). This lecture will look
at some of those techniques.
Cloning and sequencing the gene that
codes for 16S rRNA is the most
widely used method.
Nucleic acids are extracted.
The 16S rRNA genes are amplified and
cloned.
The genes are sequenced.
The sequence is identified using
phylogenetic software.
If we use DNA extracts from microbial
communities, the cloning step has to be included.
This is needed to separate the different copies of
16S rDNA. A mixed template cannot be
sequenced.
There are over 240,000 sequences deposited in
the 16S rDNA NCBI-database.
Half belong to non-cultured and unknown
organisms, which were found by 16S rDNA
cloning.
Cloning takes lots of time and so it is
not good for analyzing larger sets of
samples.
For example, it is not good for looking
for changes in natural or engineered
microbial communities over time.
Outline of the cloning procedure for
studying a microbial community.
(A) Direct nucleic acid extraction,
without the need for previous
isolation of microorganisms.
(B) amplification of the genes that code
for 16S rRNA by polymerase chain
reaction (PCR), commonly using
universal primers for bacteria or
archaea
(C) cloning of the PCR
products into a suitable
plasmid and
transformation of E. coli
cells with this vector
(E) selection of transformed clones with
an indicator contained in the plasmid
(the white colonies) and extraction of
plasmid DNA
(F) sequencing of the cloned gene,
creating a clone library
(G) Finding the relationships between
the cloned sequences of the
organisms with the help of computer
programs and databases
http://rdp.cme.msu.edu/
The Ribosomal Database Project (RDP)
provides ribosome related data and
services to the scientific community,
including online data analysis and aligned
and annotated Bacterial small-subunit 16S
rRNA sequences.
Cloning Advantages
Complete 16S rRNA sequencing allows:
very precise taxonomic studies and phylogenetic trees
of high resolution to be obtained;
design of primers (for PCR) and probes (for FISH).
If time and effort is available, the approach covers
most microorganisms, including minority groups,
which would be hard to detect with genetic
fingerprinting methods.
Cloning Disadvantages
Very time consuming and laborious, making it
unpractical for high sample throughput.
Extraction of a DNA pool representative of the
microbial community can be difficult when
working with certain sample types (e.g. soil,
sediments).
Many clones have to be sequenced so that most of
individual species in the sample are covered.
Identification of microorganisms that have not
been yet cultured or identified is difficult.
It is not quantitative. The PCR step can favor
certain species due to differences in DNA target
site accessibility.
Examples of use of clones
Examples of the use of cloning
Find the phylogenetic position of filamentous bacteria
in granular sludge.
Find the prevalent sulfate reducing bacteria in a biofilm.
The microbial communities residing in reactors for
treating several types of industrial wastewater.
The microbial composition and structure of a rotating
biological contactor biofilm for the treatment of
ammonium-contaminated wastewaters.
A description of the microbial communities
responsible for the anaerobic digestion of manure in
continuously stirred tank reactors (CSTR)
Environmental Whole-Genome Amplification To Access Microbial
Populations in Contaminated Sediments
• Recovery of adequate amounts of DNA for molecular analyses can often be
challenging in stressed microbial environments.
• Developed multiple displacement amplification (MDA) methods for unbiased,
isothermal, amplification of DNA
• Subsequently applied these technologies to understand stressed, low biomass,
populations in multiple sediments contaminated with Uranium on the Oak Ridge
Reservation
• Over 4000 clones were end sequenced. 5% of all clones were identified as
belonging to Deltaproteobacteria (primarily, Geobacter and Desulfovibrio-like)
• Significant overabundance of proteins (COGs) associated with: 1) Carbohydrate
transport & metabol. 2) Energy production & conversion, 3) Postranslational
modification, protein turnover, & chaperones. --- All of which may be important in
adaptation to environmental stressors such as low pH, high contaminate loads, and
oligotrophic nature of the subsurface environment
Library
Statistics on amplified metagenome library end-sequences
Area 3,
Area 3,
Area 2
Shallow
Deep
%
%
960
864
864
1,920
1,728
1,728
1,394
100
1,118
100
1,509
370
26.5
152
13.6
141
101
53
54
928
66.6
692
61.9
990
901
64.6
629
56.3
890
35
2.5
23
2.1
155
12
0.9
43
3.8
79
12
0.9
18
1.6
21
Number of clones sequenced
Sequences generated
a
Quality sequences
Sequences that form contigs
Number of contigs assembled
b
Sequences with similarities to known proteins
Highest similarity to bacterial proteins
Highest similarity to Deltaproteobacteria proteins
Highest similarity to archaeal proteins
Highest similarity to eukaryotic proteins
a. Sequences >400nt in length
b. e-values <1e-10 from BLASTX searches against the NCBI protein database
Abulencia, C.B., Wyborski, D.L., Garcia, J., Podar, M., Chen, W., Chang, S. H.,
Chang, H.W., Watson, D., Brodie, E.L., Hazen, T.C. and Keller, M. (2006)
Environmental Whole-Genome Amplification to Access Microbial Populations in
Contaminated Sediments. Appl. Environ. Microbiol. 72(5):3291-3301 [download
pdf]
%
100
9.3
65.6
59.0
10.3
5.2
1.4
Total
%
4,021
663
208
2,610
2,420
213
134
51
100
16.5
64.9
60.2
5.3
3.3
1.3
Metagenomic Analysis of NABIR FRC
Groundwater Community
Data: Jizhong Zhou et al.
Metagenomic sequencing:
Almost like a mono-culture
52.44 Mb raw data assembled into contigs totaling
~5.5 Mb
224 scaffolds (largest 2.4 Mb)
Genes important to the survival and life style in such
environment were found
Extremely low diversity:
Dominated by Frateuria-like organism
At least 2 Frateuria phylotypes
Azoarcus species: less abundant
These results suggest that contaminants have
dramatic effects on the groundwater microbial
communities, and these populations are well adapted to
such environments.
Frateuria 99%
Herbaspirillum 99%
Alcaligenes 98%
Frateuria 100%
Frateuria 96%
Frateuria 95%
Burkholderia 99%
Frateuria 96%
Burkholderia 99%
Frateuria 98%
Phylogenetic Tree of SSU rRNA Genes
•Four major groups were
observed.
•These microorganisms
were also found in other
studies in this site
Data: Jizhong Zhou et al.
Terry Hazen et al.
BFXI386
AY622233 NABIR FRC soil clone --Reardon
DQ125888 NABIR FRC soil clone --Brodie
FRC Gamma Group I (87.4%)
4000601 Contig2585 16SrRNA
DQ125806 NABIR FRC soil clone --Brodie
AY218719 Uncultured bacterium clone KD78
AY218686 Uncultured bacterium clone KD81
AY188295 Uncultured bacterium clone KD11
AJ010481 Frateuria aurantia
AY495957 Frateuria WJ64
AB100608 Rhodanobacter fulvus
AF039167 Rhodanobacter lindaniclasticus
L76222 Rhodanobacter lindaniclasticus
BFXI433
AJ583181 uncultured russian disposal site clone
DQ125572 NABIR FRC soil clone --Brodie
FRC Gamma Group II (1.6%)
OR1-87 NABIR FRC soil isolate --Bollmann
DQ125555 NABIR FRC soil clone --Brodie
OR1-92 NABIR FRC soil isolate --Bollmann
OR1-113 NABIR FRC soil isolate --Bollmann
BFXI385
AM084888 uranium mining waste pile clone
AJ012069 Herbaspirillum G8A1
FRC Beta Group II (4.7)
AJ505863 Herbaspirillum sp PIV341
Y10146 Herbaspirillum seropedicae
AF164065 Herbaspirillum seropedicae
BFXI398
AY662003 NABIR FRC groundwater clone --Fields
AF408965 Burkholderia str. Ellin123
FRC Beta Group I (3.1%)
AF408997 Burkholderia str Ellin155
AF408977 Burkholderia str Ellin135
AF408962 Burkholderia str Ellin120