* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Advanced Environmental Biotechnology II
Agarose gel electrophoresis wikipedia , lookup
DNA sequencing wikipedia , lookup
Gene expression profiling wikipedia , lookup
Maurice Wilkins wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene expression wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Genome evolution wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Silencer (genetics) wikipedia , lookup
DNA vaccination wikipedia , lookup
DNA supercoil wikipedia , lookup
Restriction enzyme wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Non-coding DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Molecular evolution wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Molecular cloning wikipedia , lookup
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