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| − | Our goal for this project is to genetically engineer E. coli bacteria that can break down plastic. These bacteria could have many different applications, such as: degrading plastic waste from labs that cannot be recycled, being used in a filter to catch and degrade micro plastic fibers from laundry, and breaking down plastic in a marine environment into harmless molecules. We made a lot of progress last year, and this year we plan to build on that progress.
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| − | Our goal for this project is to genetically engineer E. coli bacteria that can break down plastic. These bacteria could have many different applications, such as: degrading plastic waste from labs that cannot be recycled, being used in a filter to catch and degrade micro plastic fibers from laundry, and breaking down plastic in a marine environment into harmless molecules. We made a lot of progress last year, and this year we plan to build on that progress.
| + | Baltimore Bio-Crew tested went through the protocols for generating the Interlab data. However, we ran into troubles because of the limitations of the instruments at the community lab we work in. The interlab data analysis it is necessary to generate data in a reproducible manner comparing absorbance data to determine cell density to Fluorcine readings. We had to adapt the protocol during the Abs600 readings because our plate reader does not have the ability to read Abs600 due to lack of an appropriate filter, although we do have the filters to read fluoricine. Instead, we manually took replicates only of each of the samples with our spectrophotometer, which is a BioPharmacia Ultraspec 2000. We were unaware of the limitations of our instrument prior to setting up the InterLab experiment so we were of limited resources and time to adapt. Due to this, we were unable to do more replicates at the time of experiment and only took two replicates for each sample instead of the four replicates. We were able to learn a lot during the process of acquiring this data and following the protocols of the Interlab but we are regretful that our data will not be useful for iGEM and to fulfill the requirement for the bronze medal. To attempt to fulfill requirements and contribute to iGEM biobrick parts we assessed the top ten coding parts in Well Documented Parts and Frequently Used Parts. This information can be found on our <a href="https://2017.igem.org/Team:Baltimore_Bio-Crew/Contribution"><u>Contributions page</u></a> and our <a href="https://2017.igem.org/Team:Baltimore_Bio-Crew/Collaborations"><u>Collaborations page.</u></a> |
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| − | While searching for solutions to the issue of plastic pollution in the Baltimore Inner Harbor, we found a paper by Yoshida et. al. describing a bacteria called Ideonella sakaiensis that was capable of degrading PET plastic into monomers. The bacteria used the enzyme PETase (chlorogenate esterase) to break down PET into MHET, and the enzyme MHETase (Lipase) to break down MHET into ethylene glycol and therephthalic acid. We decided to use the genes from this bacteria for our project.
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| − | To avoid the safety risks of working with a relatively undocumented bacteria, we decided to take the plastic degradation genes from I. sakaiensis and put them into K12 E. coli bacteria. We chose E. coli because they are safe to work with and commonly used in the lab. Using the genetic sequence found in the paper, we designed the two plastic degrading enzymes so that they could be expressed in E. coli bacteria. We then had them synthesized and worked on putting these genes into E. coli.
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| − | By the end of last year’s competition, we had managed to insert the lipase gene into E.coli, but not the chlorogenate esterase gene. We confirmed that we had correctly inserted the lipase gene using colony PCR and gene sequencing, but we did not have the time to conduct additional assays, such as protein gels, to determine if the enzyme was being secreted from the bacteria. This year, we plan to redesign the chlorogenate esterase and lipase genes so that they contain the proper tags that will allow them to be detected, and a secretion sequence. After we insert both genes into E. coli cells, we will test them to make sure they can secrete the plastic degrading enzymes and degrade PET plastic.
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