Difference between revisions of "Team:UMaryland/Improve"

One of the factors that affects the efficiency of biodegradation of plastics is the concentration of the catalyzing enzyme near the surface of the plastic. If the enzyme is allowed to diffuse in solution freely it is less likely to interact with the plastic. In a situation like this degradation could be much slower than if the enzyme was highly concentrated around the surface of the plastic. The Ideonella sakaiensis was reported to have grown in a biofilm which enveloped plastic pieces. As such, we first considered maximizing production of biofilm on plastic. While researching this possibility we found that the 2017 ITB Indonesia iGEM team had attempted a similar project. They developed a system that facilitated biofilm production and expressed PETase simultaneously. Their results indicated that biofilm formation actually inhibited degradation. More plastic was degraded without biofilm than with.

We decided to investigate other methods of localizing concentration while simultaneously allowing for the greater interaction of the substrate and enzyme than biofilm was seen to do. Imperial College of London in 2014 developed parts for a cellulose binding domain (CBD). This domain was capable of being incorporated with other subunits such as GFP. They were able to demonstrate that a GFP + CBD fusion protein was capable of binding to bacterial cellulose. Equipped with this information we decided to develop a fusion protein which incorporated PETase and CBD. We assembled this part, and its characterization is available on the <a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K2825000"><u>registry</u></a>. Our hypothesis is that the bacterial cellulose surrounding the plastic will act as a scaffold on which the PETase can easily work in close proximity to the substrate. This should, in turn, increase the degradation rate. A model of our predictions can be found on the modeling page.