Team:Baltimore Bio-Crew/Design


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BALTIMORE BIO-CREW

Bio-Engineering E.Coli To Degrade Plastic and Save The Baltimore Inner Harbor

Design

This project started with only a few things in mind. Our team members knew they wanted to find a solution to an environmental issue that is prevalent in Baltimore City, and they wanted to develop a solution that could be created using the tools in the lab.
While researching solutions to the issue of plastic pollution, we found a paper describing a bacteria that could degrade PET plastic. This bacteria is known as Ideonella sakaiensis.
Found in Yoshida Sasuke's Lab, the bacterium Ideonella s. degraded the surface of a thin PET film at a rate of 0.13 mg/cm2/day at an incubation temperature of 30°C.
Two enzymes that were secreted onto film were believed to have aided in the almost complete degradation of the PET film. These enzymes are:
PETase (Chlorogenate Esterase) :
  • catalyzes PET hydrolysis
  • Turns PET into MHET (Mono (2-hydroxyethyl) Terephthalate acid)
MHETase (Lipase) :
  • Breaks down MHET
  • Turns MHET into ethylene glycol and terephthalic acid.
Ethylene glycol and Terephthalic acid are used as energy source for I. sakaiensis.
We considered using the I. sakaiensis bacteria itself, but we decided that working with a newly discovered and relatively undocumented bacteria was too risky. So we had to find a safe and effective way of recreating something with the abilities of I. sakaiensis.
Instead of using the Ideonella sakaiensis bacteria to degrade plastic, we decided to insert the genes for plastic degradation into K-12 E.coli bacteria. We chose K-12 E. coli because it is safe to use, easily grown in the lab, and very well documented since it is commonly used.

Our Constructs

After obtaining the E.coli, we designed a construct, known as a biobrick, to allow the bacteria to express the proteins lipase and esterase that will degrade PET plastics.
First is the Lac Regulated Promoter, which starts transcription in the presence of lactose or IPTG. We chose a lac regulated promoter because we wanted to have better control over the expression of the two enzymes.
Secondly, we had the Ribosome Binding site which signals the start of translation from mRNA to protein.
During the first year of our project, the design of our constructs was a bit lacking. This year, we made improvements to our biobricks that would allow them to function better. To make sure the two enzymes the bacteria produced would be able to exit the cell, we added a PelB periplasmic secretion tag. Once the enzymes were secreted to the periplasm, they would be able to exit the cell on their own.
After the PelB tag came the gene for plastic degradation. One construct had esterase, and one construct had lipase.
To allow us to easily isolate the enzymes once they were produced, we added a his tag to our constructs. This would allow us to purify our enzymes on a nickel column before running them on a protein gel. These design changes would contribute to the success of our project by allowing the enzymes to be secreted from the cell and allowing us to easily test for enzyme production.
Finally, our construct contains a terminator that STOPS transcription of the gene sequence.