1. Primary Metabolism

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

3. Loading Module Module 2 Module 3 Module 4 Module 5 Module 6 Module 7 Chemical Genetics Secondary metabolite Genes (Open Reading Frames) Gene Cluster AT ACPACP KS ACPACP KR m ATKS ACPACP KR m ATKS ACPACP m ATKS ACPACP m AT ER DHKR KS ACPACP KR ATKS ACPACP KR m ATTE Domains DNA sequence

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

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

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

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

8. Project #1: Operon/Cluster identification tool 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 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.

9. Identifying Gene Clusters 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 BLAST vs contig sequences Evaluate proximity of hits Examine predicted function of genes around “clusters” of hits. Determine boundaries for putative clusters DNA sequence(s) Putative clusters The hard part NP Chemistry Biochemistry Enzymology Automation?

11. Genome Library of query genes and desirability scores Gene extract Score for each gene 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 Low scores DNA sequence(s) Putative clusters Genome Genome(s) Map across chromosome

12. Project #2: Synthetic Sequence Generator 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 Rationale: Synthesis of large DNA sequences is commercially feasible. There is need for an organism-agnostic design tool.

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

14. Old Way Dan’s Way Type a DNA sequence Email sequence to a company to synthesize it Transform into expression host Culture organism Evaluate activity in vivo 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 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 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 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 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.

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