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Plan A Topics? 1. 2. 3. 4. 5. 6. 7. Bypassing Calvin cycle Making vectors for Dr. Harms Making vectors for Dr. Lucent Cloning & sequencing antisense RNA Studying ncRNA Engineering biodiesel, hydrogen or electricity production. Something else? Plan A http://biophotovoltaics.wordpress.com/ Photosynthetic biofilms in pure culture harness solar energy in mediatorless bio-photovoltaic cell (BPV) system. Here we report on light-driven electrical power generated with biofilms grown from photosynthetic fresh water or marine species without the addition of an artificial electron-shuttling mediator. Green alga (Chlorella vulgaris, Dunaliella tertiolecta) or cyanobacteria (Synechocystis sp. PCC 6803, Synechococcus sp. WH 5701) strains were grown directly on a transparent, conductive anode (indium tin oxide-coated polyethylene terephthalate) and power generation under light and dark conditions was evaluated using a single-chamber bio-photovoltaic cell (BPV) system. Increased power outputs were observed for all strains upon illumination, with the largest light effect observed for Synechococcus (maximum 10.3 mW m−2 total power output recorded under 10 W m−2 white light). Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA 5) Sequencing Genome projects 1) Prepare map of genome Genome projects 1) Prepare map of genome • To find genes must know their location Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library FISH to find their chromosome Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library • FISH to find their chromosome • identify overlapping AC using ends as probes • assemble contigs until chromosome is covered Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library 4) Subdivide each AC into lambda contigs Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library 4) Subdivide each AC into lambda contigs 5) Subdivide each lambda into plasmids 6) sequence the plasmids Using the genome Studying expression of all genes simultaneously Microarrays (reverse Northerns) •Attach probes that detect genes to solid support Using the genome Studying expression of all genes simultaneously Microarrays (reverse Northerns) •Attach probes that detect genes to solid support •cDNA or oligonucleotides •Tiling path = probes for entire genome Microarrays (reverse Northerns) •Attach probes that detect genes to solid support •cDNA or oligonucleotides •Tiling path = probes for entire genome •Hybridize with labeled targets Microarrays •Attach cloned genes to solid support •Hybridize with labeled targets •Measure amount of target bound to each probe Microarrays Measure amount of probe bound to each clone Use fluorescent dye : can quantitate light emitted Microarrays Compare amounts of mRNA in different tissues or treatments by labeling each “target” with a different dye Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” • Fix probes to slide at known locations, hyb with labeled targets, then analyze data Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing Using the genome Studying expression of all genes simultaneously 1. Microarrays: “reverse Northerns” 2. High-throughput sequencing • “Re-sequencing” to detect variation Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing •“Re-sequencing” to detect variation •Sequencing all mRNA to quantitate gene expression Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing •“Re-sequencing” to detect variation •Sequencing all mRNA to quantitate gene expression •Sequencing all mRNA to identify and quantitate splicing variants Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing •“Re-sequencing” to detect variation •Sequencing all mRNA to quantitate gene expression •Sequencing all mRNA to identify and quantitate splicing variants •Sequencing all RNA to identify and quantitate ncRNA Using the genome Studying expression of all genes simultaneously 1.Microarrays: “reverse Northerns” 2.High-throughput sequencing 3. Bisulfite sequencing to detect C methylation Using the genome Bisulfite sequencing to detect C methylation Using the genome Bisulfite sequencing to detect C methylation ChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells Using the genome • various chromatin modifications are associated with activated & repressed genes •Acetylation, egH3K9Ac, is associated with active genes Using the Genome •various chromatin modifications are associated with activated & repressed genes •Acetylation, egH3K9Ac, is associated with active genes • Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation Using the Genome •Acetylation, egH3K9Ac, is associated with active genes • Phosphorylation shows condensation • Ubiquitination of H2A and H2B shows repression & marks DNA damage Using the Genome •Acetylation, egH3K9Ac, is associated with active genes • Phosphorylation shows condensation • Ubiquitination of H2A and H2B shows repression • Methylation is more complex: H3K36me3 = on •H3K27me3 = off Using the Genome Methylation is more complex: •H3K36me3 = on •H3K27me3 = off •H3K4me1 = off •H3K4me2 = primed •H3K4me3 = on Using the genome Many sites provide gene expression data online • NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data Using the genome Many sites provide gene expression data online • NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data •Many different sites provide “digital Northerns” or other comparative analyses of gene expression • http://cgap.nci.nih.gov/SAGE • http://www.weigelworld.org/research/projects/geneexpr essionatlas Using the genome Many sites provide gene expression data online • NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data •Many different sites provide “digital Northerns” or other comparative analyses of gene expression • http://cgap.nci.nih.gov/SAGE • http://www.weigelworld.org/research/projects/geneexpr essionatlas • MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/ Using the genome Many sites provide gene expression data online Many sites provide other kinds of genomic data online • http://encodeproject.org/ENCODE/ Primer/probe design Crucial for successful DNA & RNA analysis! • Main source of specificity for PCR Using the genome Studying specific genes identified by WEB search • Using PCR to focus on specific genes/conditions Primer/probe design Crucial for successful DNA & RNA analysis! • Main source of specificity for PCR •good primers only bind your sequence Primer/probe design Crucial for successful DNA & RNA analysis! • Main source of specificity for PCR •good primers only bind your sequence • Also important for microarrays, sequencing, Southerns Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity: only want them to bind at one place Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity: only want them to bind at one place •Main concern: 3’ end should not bind Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: •Hairpins: may not melt (problem for RT) or may reform Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: •Hairpins • homoduplexes may not melt May be extended by DNA polymerase Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: •Hairpins • homoduplexes • heteroduplexes may not melt May be extended by DNA polymerase Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: • Melting T • Should match! Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: • Melting T • Should match! • Every site calculates them differently! Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: • Melting T • Targeting specific locations • amplifying specific sequences Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: • Melting T • Targeting specific locations • amplifying specific sequences • creating mutations: need mismatch towards 5’ end so 3’ end binds well Primer/probe design • Also important for microarrays, sequencing, Southerns • Concerns •Specificity • Complementarity: • Melting T • Targeting specific locations • amplifying specific sequences • creating mutations: need mismatch towards 5’ end so 3’ end binds well •Add restriction sites at 5’ end: may need to reamplify an amplicon