Advanced Environmental Biotechnology II

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Advanced Environmental Biotechnology II

• Week 14 - Gene cloning - gene libraries and the
  selection of clones

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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.

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• 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.

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• 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.

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• 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.

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• 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 GC 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.

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• Molecular approaches for microbial community
  analysis

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• Molecular approaches for microbial community
  analysis

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• 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.

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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.

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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.

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Restriction Enzymes

• 3. Examples of the DNA sequences that are
  recognized by other restriction enzymes are shown
  below.
• HaeIII TaqI
• 5 G G C C 3 5 T C G A 3
• 3 C C G G 5 3 A G C T 5
• PstI
  NotI
• 5 C T G C A G 3 5 G C G G C C G C
  3
• 3 G A C G T C 5 3 C G C C G G C G
  5

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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.

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DNA Ligase

• DNA ligase is an enzyme that can join (ligate)
  DNA molecules together.
• Restriction enzymes and DNA ligase are used to
  clone DNA.

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Cutting and ligating DNA
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• Strategies and steps in cloning.

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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.

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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.

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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.

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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).

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• 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

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http//www.whfreeman.com/lodish4e/con_index.htm?99
vos
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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.

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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.

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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.

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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.

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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

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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

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• 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.

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• We dont know enough about the needs of
  microorganisms.
• We dont know enough about the relationships
  between organisms.
• So we cant 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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Outline of the cloning procedure for studying a
microbial community.
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(A) Direct nucleic acid extraction, without the
need for previous isolation of microorganisms.
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• (B) amplification of the genes that code for 16S
  rRNA by polymerase chain reaction (PCR), commonly
  using universal primers for bacteria or archaea

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• (C) cloning of the PCR products into a suitable
  plasmid and transformation of E. coli cells with
  this vector

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• (E) selection of transformed clones with an
  indicator contained in the plasmid (the white
  colonies) and extraction of plasmid DNA

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• (F) sequencing of the cloned gene, creating a
  clone library

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• (G) Finding the relationships between the cloned
  sequences of the organisms with the help of
  computer programs and databases

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• 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.

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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.

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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.

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• Examples of use of clones

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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)

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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

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
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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.
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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.