Difference between revisions of "Team:Baltimore Bio-Crew/Results"

 
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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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Working hard, we managed to successfully clone the Esterase gene into E.coli. Both colony PCR and sequencing were positive for the CE 5 esterase clone. However, Lipase was not successfully cloned and the sequences came up negative and truncated.</article>
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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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Due to the negative results with the lipase, we hypothesized that the lipase enzyme was toxic to the strain of E. coli we were using, causing it to reject the DNA.
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We started inducing the positive esterase clone to produce protein, and running the protein produced on a protein gel after purifying on a nickel column. After running multiple protein gels with the esterase, no positive results were found.
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Because of this, we hypothesized that the E. coli cells were not producing the esterase because the enzyme was also harmful to the cells.
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<img src="https://static.igem.org/mediawiki/2017/5/5e/T--Baltimore_Bio-Crew--results_fig2.jpg" style="width:960px; height: 540px;"><!--original size: 1,920 × 1,080 pixels-->
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To bypass the cell issue and test whether or not our constructs could actually produce the protein, we decided to test the constructs and biobricks on their own by translating them using a cell free kit, and then running the product of that on a protein gel. The protein gel results after the cell free kit were inconclusive, possibly because the cell just wasn’t producing enough protein to show up on the gel.
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<img src="https://static.igem.org/mediawiki/2017/e/ec/T--Baltimore_Bio-Crew--results_fig3.jpg" style="width:960px; height: 540px;"><!--original size: 1,920 × 1,080 pixels-->
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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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We decided to run the cell free kit products through a FDA enzyme test. Fluorescence was seen, indicating that enzyme was being produced! The results of this test are shown on a bar graph below
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Additional tests will need to be done, but the FDA test indicated that our constructs can produce protein, even if the enzymes may not be able to be produced in E. coli
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   <img src="https://static.igem.org/mediawiki/2016/6/65/T--Baltimore_BioCrew--DeutschFoundation.png" alt="The Robert W. Deutsch Foundation" style="width:100px; height:100px;">
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   <img src="https://static.igem.org/mediawiki/2017/6/6c/T--Baltimore_Bio-Crew--fabian_kolker_small_icon.png" alt="Fabian Kolker Foundation" style="width:100px; height:100px;">
 
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<a href="http://www.marylandrecyclingnetwork.org/">
 
   <img src="https://media.licdn.com/mpr/mpr/shrink_200_200/AAEAAQAAAAAAAAI8AAAAJDY0ZDg0ZjlkLWVlMTItNGI1Mi1iNWEwLWYzMDVlYWMwMTZhZg.png" alt="Maryland Recycling Network" style="width:100px; height:100px;">
 
   <img src="https://media.licdn.com/mpr/mpr/shrink_200_200/AAEAAQAAAAAAAAI8AAAAJDY0ZDg0ZjlkLWVlMTItNGI1Mi1iNWEwLWYzMDVlYWMwMTZhZg.png" alt="Maryland Recycling Network" style="width:100px; height:100px;">
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<a href="https://www.rwdfoundation.org/">
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  <img src="https://static.igem.org/mediawiki/2016/6/65/T--Baltimore_BioCrew--DeutschFoundation.png" alt="The Robert W. Deutsch Foundation" style="width:100px; height:100px;">
 
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Latest revision as of 19:40, 19 November 2017


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BALTIMORE BIO-CREW

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

Results

Working hard, we managed to successfully clone the Esterase gene into E.coli. Both colony PCR and sequencing were positive for the CE 5 esterase clone. However, Lipase was not successfully cloned and the sequences came up negative and truncated.
Due to the negative results with the lipase, we hypothesized that the lipase enzyme was toxic to the strain of E. coli we were using, causing it to reject the DNA.
We started inducing the positive esterase clone to produce protein, and running the protein produced on a protein gel after purifying on a nickel column. After running multiple protein gels with the esterase, no positive results were found.
Because of this, we hypothesized that the E. coli cells were not producing the esterase because the enzyme was also harmful to the cells.
To bypass the cell issue and test whether or not our constructs could actually produce the protein, we decided to test the constructs and biobricks on their own by translating them using a cell free kit, and then running the product of that on a protein gel. The protein gel results after the cell free kit were inconclusive, possibly because the cell just wasn’t producing enough protein to show up on the gel.
We decided to run the cell free kit products through a FDA enzyme test. Fluorescence was seen, indicating that enzyme was being produced! The results of this test are shown on a bar graph below
Additional tests will need to be done, but the FDA test indicated that our constructs can produce protein, even if the enzymes may not be able to be produced in E. coli