The breakdown of Pet Kristen Meeh

Overview of bacteria being used Ideonella Sakaiensis Rod shaped Produces the enzymes PETase and MHETase to break down the PET Breaks the polymer down into two environmentally benign monomers Hydrolyzes PET Recycled back into the bacterium Accessory gene Pirellula (Rhodopirellula baltica) Marine bacteria Found worldwide Aerobic Heterotrophic

PET PET or PETE is short for polyethylene terephthalate fibers for clothing, containers, thermoforming for manufacturing, and in combination with glass fiber for engineering resins 60% of synthetic fabrics Bottle production accounts for 30% of global demand decomposed at a temperature of at least 350 degrees Celsius Insoluble in water

Purpose Synthetically improve Ideonella sakainesis Ideonella can only work at the temperature of 30 degrees Celsius (86 degrees Fahrenheit) Improved bacteria will be able to break down the inordinate amount of plastic in the ocean Reduce negative effects of plastic Sea life is negatively affected or even killed by plastic

Purpose Production of plastic Humans release between 5.3 million and 14 million tons of plastic annually The Great Plastic Garbage Patch (Pacific trash vortex) that spans waters from the West Coast of North America to Japan It is comprised of the Western Garbage Patch and the Eastern Garbage Patch

Competing Technologies Reduce, reuse, recycle Can not remove the preexisting plastic Makeup of plastic is harmful to the environment Need to get more people to recycle and reuse the plastic The Seabin Founded by Andrew Turton and Pete Ceglinski Pump connected to the bottom Pump uses air pressure to suck in debris and plastic which is pulled up into a container on land

Design Plasmid from Ideonella will be placed into Pirellula PET plastic will be picked up by multiple receptors Strong promoter PET used as food source for Ideonella=attracted to the plastic PCR will be used Pirellula as the chassis Ideonella plasmid inputted via PCR

Design of Bacterium and Operon PM- binding particle produced by receptors A- activator T- terminator Yellow rectangle- start codon Red rectangle- stop codon Purple hexagon- PETase Pink pentagon- MHETase Grey rectangles- receptors Brown Cross- ethylene glycol Red Raindrop- terephthalic acid + ORF RBS RNAP A PM T PETase MHETase (enzymes) PET

Expected Results Behavior of Bacterium when Working Perfectly Solution Be able to survive in a marine environment Have the gene for breaking down PET Be able to produce more enzymes when more PET is present Be attracted to the PET Solution Plastic with PET will be broken down=better environment Presence of PET Production of Enzymes 1

Advantages Monomers are recycled back into the bacterium; positive feedback

Potential Problems Speed of processing The receptors are not created The particle the receptor will produce to activate the enzyme is not determined Pirellula could produce a repressor for the operon; inhibiting the enzyme from being produced

Production Safety Protection of the Environment both bacteria are not harmful Protection of the Environment No harmful fumes or by products will be produced when synthesizing Materials will be disposed of properly

Evolution Pirellula has had few changes from evolution Pirellula formed a cell envelope; it lost some genes which coded for peptidoglycan Peptidoglycan is the formation of amino acids and sugars into a mesh-like layer around the plasma membrane Because Ideonella is a newly discovered bacteria there was no research conducted looking into its evolution

Testing Gel electrophoresis to see if the DNA from Ideonella was accepted into Pirellula If gel works the bacterium will be placed into a marine environment with PET plastics to see if the bacterium can survive in the environment and break down the PET The receptors and the production of enzymes will be the main components By products and growth rate will be monitored