Laura Branch and Roxanne Daniels Discussion Abstract Results Plastic waste harms and kills a vast amount of fish and marine Why use genes from I. sakaesis? Why aren’t mammals each year. These marine animals fall victim to death as a result other bacterial cutinase genes good enough? of ingesting, suffocation, and entanglement from plastic debris. ●Both proteins act using a “catalytic triad” Preventative measures have been taken to help fix the problem, but mechanism involving interactions between the methods to decrease the amount of plastics from reaching the ocean but have had little long term success.2 Recently, a promising new method proteins Ser, His, and Asp (the residues vary was discovered utilizing a bacterium known as Ideonella Sakaiensis that is between cutinases and PETase) able to biodegrade plastics. I.Sakaiensis is able to thrive on PET ●New studies have revealed that PETase has a (polyethylene terephthalate) as a major carbon and energy source. It is 52% similarity with the cutinase found in able to break down PETs with two enzymes PETase and MHETase.5 The purpose of this experiment is to create a microbe capable of thriving in Thermobifida fusca (this enzyme also degrades the ocean and of breaking down ocean plastics. In order to create this PET)1 plasmid, the genes that produce PETase and MHETase will first be ○ I. sakaesis’ PETase gene is a less specific isolated and then cloned into a biobrick plasmid backbone using restriction digestion. They will later be transformed into a marine species version, which makes it more effective of Bacillus and selected for by plating the transformed competent cells ○ I. sakaesis’ PETase has a deeper cleft (which on LB plates containing chloramphenicol. The cloned plasmid DNA will be can be seen in figure 2) allowing for more further analyzed through the use of gel electrophoresis and PCR. As an conformational change in the enzyme expected result, it is theorized that the experiment will be successful and Figure 1. PETase catalyzes the depolymerization of PET that the desired cloned plasmid containing the two enzymes (PETase and ○ A single amino acid substitution from to bis(2-hydroxyethyl)-TPA (BHET), MHET, and TPA. MHETase) within the biobrick plasmid backbone will be obtained. MHETase converts MHET to TPA and EG.1 phenylalanine to serine in the lining of the active site Introduction PETase vs. Other Cutinases ● Similarly, Trp159 in PETase extends the hydrophobic surface adjacent to the The accumulation of plastics in the Earth’s oceans is an increasingly Figure 2: Differences in proteins. (E) View active site urgent problem that is not receiving enough attention. The floating plastics affect all ranks of the ecosystem, from organisms as small as along the active-site cleft of PETase ○ The PETase found in I. sakaesis is covered phytoplankton all the way up to seabirds, like the albatross. corresponding to the area highlighted with a vast amount of basic amino acid Dissection of seabirds has revealed the ingestion of many plastic with a red dashed circle in A and C. The residues, compared to the more equal products, including cling wrap, popped balloons, and toothpicks. spread of basic and acidic amino acid width of the cleft is shown between Why do these birds ingest so much plastic? The answer lies in the smell of the plastic; the odor of rotting seaweed is very similar to Thr88 and Ser238. (F) Narrower cleft of residues in T. fusca cutinase. the smells emitted when plankton attack ocean plastics.2 the T. fusca cutinase active site is shown ● PETase has a pI of 9.6 compared to T. In a recycling plant in Japan, researchers stumbled upon a with the width between Thr61 and fusca cutinase’s more neutral 6.3 species of bacteria, Ideonella sakaiensis, that were able to survive by 1 ○ The sulfide bonds found in PETase’s Phe209 in equivalent positions. metabolizing polyethylene terephthalate (PET) in plastics as their C-terminus end as well as in the active site, sole food source. PET is similar to polyester and is the clear plastic used to mold bottles. After genetic analysis of I. sakaiensis, it was Materials are thought to provide stability to the protein, similar to a culture of Fusarium determined that I. sakaiensis produces two enzymes (PETase and solani’s cutinase1 MHETase) that fully break down plastic.5 The aim of this experiment is to create a microbe capable of thriving in the ocean and of breaking down ocean plastics. It is hypothesized that the cloned Conclusions plasmid containing the two enzymes will be successfully selected for and implemented into marine Bacillus. and Future Work Because PETase has less specific protein-substrate interactions, PET has been shown to break down today’s
Methods Figure 3. MHETase plasmid map Figure 4. 3
PETase plasmid map 3 alternate more biodegradable plastic, PEF, as well. With that being said, PETase has the ability to at least partially Initially, all three plasmids were digested with restriction enzyme EcoRI. To break down most aromatic plastics.1 The future of ligate the PETase and METase to the backbone, T4 DNA ligase was used PETase may include genetically engineering it to behave along with a ligation buffer using a standard ligation procedure. To transform so that it can break down aliphatic polyesters as well as into Bacillus subtilis, the cells were made competent by intermittently adding aromatics, or to create a new protein using the catalytic CaCl2 and heat shocking them in a water bath. The pSB1C3 backbone triad mechanism that can attack aliphatic polyesters contains the resistance gene to chloramphenicol. The transformed cells were specifically. then grown on two sets of plates; LB and LB + CHLO.*
*A control procedure was completed alongside without the initial digestion References with EcoRI to confirm that non-transformed cells would not grow on the LB + 1. Austin HP, Allen MD, Donohoe BS, et al. Characterization and engineering CHLO plates. Figure 5. pSB1C3 backbone Figure 6. Image of Bacillus subtilis4 of a plastic-degrading aromatic polyesterase. Proceedings of the National plasmid map3 An SDS-PAGE gel was also run to confirm uptake of the plasmid. SDS is a Academy of Sciences. 2018:201718804. doi:10.1073/pnas.1718804115. detergent that leads to the lysis of the bacterial cell wall. The sodium hydroxide raises the pH, which not only denatures cell proteins, but also denatures DNA to single strands. After running a standard DNA extraction Safety 2. Briggs H. Chemical clue to why seabirds eat plastic. BBC Science and
procedure in Hi-Bind DNA mini columns, prepared DNA from each growth The organisms used in this study are Ideonella sakaiensis and a marine strain of Environment. http://www.bbc.com/news/science-environment-37926733. plate was put into the wells of a 0.9% agarose gel and run for an hour at Bacillus subtilis, which are both biosafety level 2.3 These are considered a level 2 Published November 10, 2016. Accessed April 1, 2018. 120V. The gel was stained with gel red after running. because they are capable of being pathogenic, but are treatable and propose minimal human risk as an infection. This project will comply with the non-environmental release 3. Laun S, Scheifele L, Avidor R, et al. Baltimore BioCrew. Verification of Clone using PCR policy, because the experiment will be done in a simulated ocean laboratory environment. As far as laboratory safety is concerned, we will be wearing gloves, http://2016.igem.org/Team:Baltimore_BioCrew. Published 2016. Accessed In order to verify if the correct clone was obtained, the full length coding sequence of the backbone was input into IDT- Integrated DNA Technologies laboratory coats, and closed-toed shoes. The laboratory work bench will be cleaned April 1, 2018. PrimerQuest Tool to design custom primers. These primers will be used to and disinfected prior to experimentation. Waste will be autoclaved and discarded in detect the presence and amplify the inserts (PETase and METase) using a their appropriate containers. We will also perform sterile technique when plating and 4. The Bacterium Bacillus subtilis taken with a Tecnai T-12 TEM. Taken by polymerase chain reaction. If the two genes did successfully clone into the culturing bacteria. Allon Weiner, The Weizmann Institute of Science, Rehovot, Israel. 2006. backbone, then there will be the presence of amplified DNA. If the two genes did not successfully clone into the backbone, then there will not be In regards to biobrick safety, the enzymes we will be using to cut and ligate the parts 5. Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and amplification of the desired region of DNA. are non-toxic to humans when used correctly. The products of gene expression will not affect humans in a negative way as long as the Bacillus are handled with proper care. assimilates poly(ethylene terephthalate). Science.
