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Primary Metabolism

Primary Metabolism. Microbial life strategies. Secondary metabolites, or natural products. “Nomads”. “Settlers”. ~3 cm. ~1 mm. ~10 cm. Actinomycetes Cyanobacteria Filamentous Fungi. E. coli Salmonella Streptococcus. Settlers have to protect themselves from the Nomads. Antibiotics.

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Primary Metabolism

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  1. Primary Metabolism

  2. Microbial life strategies Secondary metabolites, or natural products “Nomads” “Settlers” ~3 cm ~1 mm ~10 cm Actinomycetes Cyanobacteria Filamentous Fungi E. coli Salmonella Streptococcus Settlers have to protect themselves from the Nomads Antibiotics Signaling molecules Siderophores Specialized cell walls/lipids

  3. Chemical Genetics DNA sequence Genes (Open Reading Frames) Gene Cluster ER KR KR DH KR KR KR Domains AT A C P KS mAT A C P KS mAT A C P KS mAT A C P KS mAT A C P KS AT A C P KS mAT A C P TE Loading Module Module 3 Module 5 Module 7 Module 2 Module 4 Module 6 Secondary metabolite

  4. The Genus Salinispora Lycopene/Iterative Type I PKS nrps2 pks4 5-module NRPS Jensen, Fenical FAS/PKS-related Cyclomarin Enediyne PKS FAS/PKS-related S. arenicola Rifamycin 5.68 Mb Lym Type I PKS NRPS-related FAS/PKS-related Calicheamycin – part1 4-module NRPS sid2 pks2 Calicheamycin – part2 FAS/PKS-related Staurosporine sid1 2-module PKS/NRPS Udwary, Ziegler, Lapidus, Moore, Jensen

  5. A Yersiniabactin Cluster? 27698 bp Yersiniabactin cluster from Yersinia pestis unknown transport

  6. The quick brown fox jumped over the lazy dog Yoda speak: Over the lazy dog the quick brown fox jumped. A fast tan dingo leaped above some sleepy mutt Low sequence identity, Similar “product” Yersiniabactin cluster from Yersinia pestis

  7. High “sequence identity”, But different “product” The quick brown fox jumped over the lazy dog The quick brown fox jumped over the lazy dog BLAST can’t really tell the difference Would lead to different, but similar, chemical products that may have different activity

  8. Project #1: Operon/Cluster identification tool Rationale: I have a need to locate conserved or related operons or gene clusters across multiple species. Important to understanding of genome evolution, and functional analysis. Goal: Develop a rapid automated method to identify conserved gene clusters or operons across genomes. Personnel needs: Programmer/scripter (Java or Perl is ideal) Biologist with some knowledge of gene structure or genomics

  9. Identifying Gene Clusters Putative clusters BLAST vs contig sequences Evaluate proximity of hits Examine predicted function of genes around “clusters” of hits. Determine boundaries for putative clusters NP Chemistry Enzymology The hard part Biochemistry Automation? PKS: KS AT DH ER KR ACP Methyl transferase MT TE NRPS: C A T (PCP) Epimerization C-cyclization N-methyltransferase TE Other: Prenyl transferase Cytochrome P450 Type III PKS KAS III DNA sequence(s)

  10. Putative clusters Genome KS AT DH ER KR ACP Methyl transferase MT TE C A T (PCP) Epimerization C-cyclization N-methyltransferase TE Prenyl transferase Cytochrome P450 KAS III High scores Genome Genome(s) Library of query genes and desirability scores extract Gene Score for each gene Map across chromosome Low scores DNA sequence(s)

  11. Project #2: Synthetic Sequence Generator Rationale: Synthesis of large DNA sequences is commercially feasible. There is need for an organism-agnostic design tool. Goal: Develop a tool to generate organism-specific DNA sequences suitable for DNA synthesis from protein sequences Personnel needs: Programmer/scripter Biologist with some knowledge of molecular biology and genetics

  12. SCO7671 - Type III PKS SCO7672 - prenyl transferase SCO7670 - Isoprenylcysteine carboxyl methyltransferase SCO7673 - lipoprotein SCO7669 - oxidoreductase SCO7668 - regulatory SCO7674 - Cu-binding plastocyanin SCO7667 - phosphohydrolase SCO7675 - unknown membrane protein S.coelicolor SCO7671 region 6901 bp STRO2878 - UbiG benzoquinol methyltransferase STRO2877 - UbiA (SCO7672) STRO2879 - Acyl-CoA dehydrogenase STRO2876 STRO2880 - Type III PKS STRO2875 - DNA repair STRO2881 STRO2874 - DNA-binding STRO2882 - transcription factor STRO2873 STRO2883 Cluster 12 - Type III region - pks4 11301 bp

  13. Problem: I want to understand what the four-gene operon from each organism does, biochemically. NEED all four proteins active and in the same place. Plus, one organism is not available to me. Dan’s Way Old Way PCR-amplify gene(s) Purify PCR product(s) Clone into expression plasmid(s) Confirm accuracy of sequence(s) Transform into expression host Culture organism Purify proteins Evaluate activity in vitro PCR-amplify gene(s) Purify PCR product(s) Clone into expression plasmid(s) Confirm accuracy of sequence(s) Transform into expression host Culture organism Purify proteins Evaluate activity in vitro PCR-amplify gene(s) Purify PCR product(s) Clone into expression plasmid(s) Confirm accuracy of sequence(s) Transform into expression host Culture organism Purify proteins Evaluate activity in vitro Type a DNA sequence Email sequence to a company to synthesize it Transform into expression host Culture organism Evaluate activity in vivo PCR-amplify gene(s) Purify PCR product(s) Clone into expression plasmid(s) Confirm accuracy of sequence(s) Transform into expression host Culture organism Purify proteins Evaluate activity in vitro

  14. Protein sequence Codon usage table of host organism Reverse translate Codon-adjusted DNA sequence Add expression tag(s) Remove restriction sites Remove or alter other undesirable motifs Optional modifications Re-engineered, optimized DNA sequence Adjust for expressible multi-gene transcript Engineered, optimized expressible DNA operon

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