UT iGEM 2012: Caffeinated coli h7p://2012.igem.org/Team:AusAn_Texas 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
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: 1417-1424. 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 http://www.geog.ucsb.edu
And who is to blame?
Source: The Huffington Post (originally from http://www.ilovecoffee.jp)
Decaffeination
•  Clean up the environmental impacts of caffeine polluAon •  Detoxify caffeine industrial waste for use as feed stock •  Produce new methylxanthine based drugs https://www.avma.org/News/PressRoom/Cows/k7964-1.jpg
http://www.sfms.org/Portals/3/assets/images/Blog/Pills.jpg
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. 194:2041-2049.
CBB5 Caffeine DemethylaAon Pathway •  CBB5 metabolizes caffeine and other methylxanthines to xanthine using an N-­‐demethylaAon pathway “Addicting” E.coli to Caffeine
PRPP IMP
inosine-5'-phosphate
GuaB
(IMP Dehydrogenase)
XMP
Xanthosine-5’-Phosphate
Xanthine
gpt
CBB5 operon
Caffeine
Xanthine salvage pathway
dGTP
DNA and RNA
Cloning naAve CBB5 demethylaAon operon -  13.2 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
experimentally proven RBSs
non-optimal promoters
experimentally proven promoters
GTG start codons
ATG start codons
uncharacterized regulators
constitutive expression
Will refactoring restore
functionality?
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)
Assembly: One-­‐step, 6-­‐piece Gibson assembly promoter = RBS (from BBa_B0034) + ATG ndmA ndmB ndmC ndmD gst9 pSB1C3 Gibson isothermal assembly
BBa_23100 promoter ndmA ndmB ndmC pSB1C3 BBa_K734000
ndmD gst9 Growth of guaB knockout with and
without decaffeination operon
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Growth in caffeinated beverages
–
+
decaffeination operon
–
+
decaffeination operon
Growth in caffeinated beverages
–
+
decaffeination operon
• 
• 
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.
Caffeine content calculations
Calculated caffeine Actual caffeine
content (mg/L)
content (mg/L)
Coca-Cola
99.1
98.6
Espresso
44.7
39.5*
* Reported value; may vary depending on preparation
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) TranscripAonal Regulators & Promoters P. pu,da naAve caffeine degradaAon operon •  Two putaAve transcripAonal regulators •  Intergenic regions may contain regulated promoters TranscripAonal Regulators & Promoters P. pu,da naAve caffeine degradaAon operon •  Two putaAve transcripAonal regulators •  Intergenic regions may contain regulated promoters TranscripAonal Regulators & Promoters P. pu,da naAve caffeine degradaAon operon •  Two putaAve transcripAonal regulators •  Intergenic regions may contain regulated promoters TranscripAonal Regulators & Promoters P. pu,da naAve caffeine degradaAon operon LacZ •  Two putaAve transcripAonal regulators •  Intergenic regions may contain regulated promoters TranscripAonal Regulators & Promoters P. pu,da naAve caffeine degradaAon operon LacZ Orf1 or Orf4 •  Two putaAve transcripAonal regulators •  Intergenic regions may contain regulated promoters Protein Based Repression of ndmA Promoter Miller Units 12000 10000 8000 6000 4000 2000 0 ndmA Promoter ndmA Promoter + ORF1 ndmA Promoter + ORF4 •  Confirms presence of ndmA promoter •  orf4 protein regulates ndmA expression TesAng for Substrate InducAon Induc9on of ndmA promoter via Methylxanthine Supplementa9on Miller Units 700 600 500 400 Caffeine 300 Theobromine 200 Theophylline 100 0 0 1 2 3 4 5 6 Supplement Concentra9on (mM) •  ndmA promoter is induced by methylxanthines 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.
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