1. The breakdown of Pet
Kristen Meeh

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. Advantages
Monomers are recycled back into the bacterium; positive feedback

11. 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

12. 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

13. 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

14. 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