Decomposition of plastics and other man-made compounds by bacterial enzymes

Noah Goldman Cluster 1: Biotechnology COSMOS 2008

Uses
Production of Compounds for industrial polymer production Bioremediation
Decomposing toxic chemicals and removing then from the environment using biological systems

Rieske non-heme iron oxygenases

Dioxygenases Oxidizes aromatic hydrocarbons by
Ex: Naphthalene, Toluene etc. naphthalene dioxygenase is decomposes over 60 different types

Found in Pseudomonas bacteria and Sphingomonads Iron center Oxidizes hydrocarbons

Chemotaxis
When bacteria are attracted to chemicals they can decompose Extremely useful for bioremediation would greatly increase efficiency Chemosensory systems follow trail to source

Plastics
Polymers Hydrocarbons Man-made As of yet, very few natural processes that can break it down

Purpose
Can I find enzymes that can decompose certain man made chemicals, such as polyurethane?
If one bacteria can decompose naphthalene, why not plastic?

Polyurethane
Complex, plastic, polymer with two primary types of molecules
Isocyanate and alcohol groups Many different things for enzymes to attach to

Collection
Go to environments with polyurethane pollution and collect dirt samples Landfills, streams near mining sites, etc

Culturing the organism

Grow the samples of bacteria from the dirt on plates where the only carbon source is the polyurethane Plate any growth to get individual colonies Sequence ribosomal DNA

Isolating the Gene

Insert transpososomes into the cells using electroportation
A transpososome is a group of custom genes which, when inserted into a cell, will bind into the genome in a random place.

Culture in media with polyurethane as the carbon source Test colonies without growth on a normal medium If the bacteria grows as well as the control on the normal media, but is showing minimal growth on the reduced media, sequence out from the transpososome to find the gene.

Producing the enzyme

PCR the identified gene and clone a His tag on Put into TOPO plasmid
lacZ Antibiotic resistance

Transformation into E. coli Culture Filter and purify protein

Testing the enzyme

Place samples of purified enzyme in a solution of radioactive polyurethane monomers and another of radioactive dimers Observe affect through paper chromatography
Run the potentially modified polyurethane and unmodified polyurethane next to each other. If one chemical moves faster/slower than the other, then it was modified, and the sample contained the first enzyme in the pathway If there was no change either wrong enzyme, or a later step in the pathway

Go back to transposition and find the other enzymes in the pathway Try different orders and mixes of enzymes

Chemicals to Test
Polyurethane Polystyrene Nylon Naphthalene Imide DDT

Materials
P2, P20, P200, P1000 pipetmen and tips LB media plates/culture tubes Media with polyurethane monomer/dimer carbon source in plates and culture tubes Polyurethane monomers and dimers with radioactive isotopes attached Transposomes TOPO plasmid Competent E. coli solution of polyurethane monomers and dimers and buffer for the enzyme HIS tag column Microscope PCR machine and mastermix Collection tubes

If Successful
I will identify the function of each enzyme in the pathway I will study the structure of the enzyme and begin modifying it to improve efficiency and durability.

If unsuccessful I will modify existing enzymes

Change an existing enzyme to break down a polyurethane. Change a biocatalyst that builds chemicals to instead break them down.

Works Cited
Aehle, Wolfgang, ed. Enzymes in Industry: Production and Applications. 2nd ed. Weinheim: Wiley-VCH, 2004. Feldstein, Paul. "Isolation of bacteria that can degrade naphthalene." COSMOS Lab wtite-up, 2004. Kauppi, Bjorn, Kyoung Lee, Et all. "Structure of an aromatic-ring-hydroxylating dioxygenase naphthalene 1,2-dioxygenase." Structure 6 (1998): 571-86. Pajunen, Maria I., Arto T. Pulliainen, Et all. "Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes." Microbiology (2005): 1209-18. Parales, Rebecca E., and David T. Gibson. "Aromatic hydrocarbon dioxygenases in environmental biotechnology." Current Opinion in Biotechnology 11 (2000): 236-43. Parales, Rebecca E., and John D. Haddock. "Biocatalytic degradation of pollutants." Current Opinion in Biotechnology 15 (2004): 374-9. Parales, Rebecca E. "The role of active-site residues in naphthalene dioxygenase." Journal of Industrial Microbiology and Biotechnology 30 (2003): 271-8. Senese, Fred. "Chromatography." General Chemistry Online! 25 July 2005.

Images Cited
Dr. Jakubowski. "CHEM 123 GENERAL CHEMISTRY 1." Online Study Guide. 30 July 2008 http://employees.csbsju.edu/hjakubowski/classes/ch123/chromatographypens.jpg. Pajunen, Maria I., Arto T. Pulliainen, Et all. "Generation of transposon insertion mutant
libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes. Microbiology (2005): 1209-18.

30 July 2008 <http://antoine.frostburg.edu/chem/senese/101/matter/ chromatography.shtml>. 30 July 2008 http://www.elmhurst.edu/~chm/onlcourse/chm110/outlines/ images/polyurethane.gif.