UT igem 2012: Caffeinated coli. h7p://2012.igem.org/team:ausan_texas
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1 UT igem 2012: Caffeinated coli h7p://2012.igem.org/team:ausan_texas
2 Caffeine Caffeine is 1,3,7- trimethylxanthine Xanthine is a guanine precursor Used in many foods, beverages, and pharmaceuacals Caffeine 120,000 tons/year made by humans Xanthine
3 Environmental Impact Most organisms cannot efficiently degrade caffeine Significant accumulaaon in the environment from industrial and waste water sources Caffeine levels are used to measure human impact on bodies of water Rodriguez et al. (2012) Marine Pollution Bulletin, 64:
4 Environmental Impact Most organisms cannot efficiently degrade caffeine Significant accumulaaon in the environment from industrial and waste water sources Caffeine levels are used to measure human impact on bodies of water
5 And who is to blame? Source: The Huffington Post (originally from
6 Decaffeination Clean up the environmental impacts of caffeine polluaon Detoxify caffeine industrial waste for use as feed stock Produce new methylxanthine based drugs
7 Pseudomonas pu,da CBB5 Discovered by Ryan Summers and Mani Subramanian at the University of Iowa Can live on caffeine as the sole carbon and nitrogen source Summers RM et al. (2012) J. Bact. 194:
8 CBB5 Caffeine DemethylaAon Pathway CBB5 metabolizes caffeine and other methylxanthines to xanthine using an N- demethylaaon pathway
9 Addicting E.coli to Caffeine PRPP IMP inosine-5'-phosphate GuaB (IMP Dehydrogenase) CBB5 operon Xanthine gpt XMP Xanthosine-5 -Phosphate Caffeine Xanthine salvage pathway dgtp DNA and RNA
10 Cloning naave CBB5 demethylaaon operon kb known sequence - Cloned into pacyc184 plasmid - Transformed into guab knockout No growth Possible incompatibilities: P. putida CBB5 optimizing for E. coli non-optimal RBSs non-optimal promoters GTG start codons uncharacterized regulators experimentally proven RBSs experimentally proven promoters ATG start codons constitutive expression Will refactoring restore functionality?
11 Refactored demethylaaon operon Promoter: (BBa_J23100) RBS: (BBa_B0034) AAAGAGGAGAAA ndmabcd refactored from P. pu,da CBB5, including ATG replacements gst9: Janthinobacterium Marseille orf8 (partial sequence)
12 Assembly: One- step, 6- piece Gibson assembly promoter ndma ndmb ndmc = RBS (from BBa_B0034) + ATG ndmd gst9 psb1c3 Gibson isothermal assembly BBa_23100 promoter ndma ndmb ndmc ndmd gst9 psb1c3 BBa_K734000
13 Growth of guab knockout with and without decaffeination operon!#$"!#($"!#("!#'$"!"#$#%!#'"!#&$"!#&"!#%$"!#%","-./0-1"2"!34"53..6/7/18" 2"-./0-1"2"!34"53..6/7/18","-./0-1"2"%!!34"9:18;<1/" 2"-./0-1"2"%!!34"9:18;<1/","-./0-1"2"%!!34"=:>/<1/" 2"-./0-1"2"%!!34"=:>/<1/"!#!$"!"!" &" (" )" *" %&" %(" %+" %*" &%" &(" &'()*%
14 Growth in caffeinated beverages + decaffeination operon + decaffeination operon
15 Growth in caffeinated beverages + decaffeination operon
16 Growth of guab knockout E.coli with decaffeination operon shows linear dependence on caffeine concentration. 7.6 ± 0.8 pg caffeine required per E. coli cell Beyond 250 µm, growth is saturated, apparently because another nutrient becomes limiting.
17 Caffeine content calculations Calculated caffeine content (mg/l) Actual caffeine content (mg/l) Coca-Cola Espresso * * Reported value; may vary depending on preparation
18 TranscripAonal Regulators & Promoters SyntheAc biology needs more inducible promoters! IPTG is expensive (>$10.00/g) DegradaAon operon is likely regulated by methylxanthines in Pseudomonas pu,da CBB5. Collins et al. SyntheAc gene networks that count. Science, 2009 Caffeine is cheap ($0.30/g)
19 TranscripAonal Regulators & Promoters P. pu,da naave caffeine degradaaon operon Two putaave transcripaonal regulators Intergenic regions may contain regulated promoters
20 TranscripAonal Regulators & Promoters P. pu,da naave caffeine degradaaon operon Two putaave transcripaonal regulators Intergenic regions may contain regulated promoters
21 TranscripAonal Regulators & Promoters P. pu,da naave caffeine degradaaon operon Two putaave transcripaonal regulators Intergenic regions may contain regulated promoters
22 TranscripAonal Regulators & Promoters P. pu,da naave caffeine degradaaon operon LacZ Two putaave transcripaonal regulators Intergenic regions may contain regulated promoters
23 TranscripAonal Regulators & Promoters P. pu,da naave caffeine degradaaon operon LacZ Orf1 or Orf4 Two putaave transcripaonal regulators Intergenic regions may contain regulated promoters
24 Protein Based Repression of ndma Promoter Miller Units ndma Promoter ndma Promoter + ORF1 ndma Promoter + ORF4 Confirms presence of ndma promoter orf4 protein regulates ndma expression
25 TesAng for Substrate InducAon 700 Induc9on of ndma promoter via Methylxanthine Supplementa9on Miller Units Caffeine Theobromine Theophylline Supplement Concentra9on (mm) ndma promoter is induced by methylxanthines
26 Summary of Results Refactored decaffeination operon from P. putida CBB5 into E. coli. Addicted" E. coli to caffeine. Used the addicted cells to accurately measure caffeine content of common beverages. Characterized a methylxanthineresponsive transcriptional regulator.
27 UT Austin igem 2012 Undergraduates Ben Slater Peter Outopal Razan Alnahhas Logan Bachman Aurko Dasgupta Will Darden Advisors Dr. Jeff Barrick Michael Hammerling Erik Quandt Jared Ellefson
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