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

 
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         <link href="https://fonts.googleapis.com/css?family=Saira|Bubbler+One|Abel|Source+Sans+Pro|Share+Tech+Mono|Kalam" rel="stylesheet">
 
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              <h2>COMPOSITE PARTS</h2>
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        <h3>Composite Parts</h3>
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We designed two constructs, known as biobricks, to allow the bacteria to express the proteins lipase and esterase that will degrade PET plastics. We designed two constructs, identical except for the sequence for the enzyme each construct would produce: esterase and lipase. Our constructs were designed as follows.
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We designed two constructs, known as biobricks, to allow the bacteria to express the proteins lipase and esterase that will degrade PET plastics. These constructs are identical except for the sequence for the enzyme each construct would produce: esterase and lipase. The chlorogenate esterase construct (BBa_K2436001) was cloned successfully and submitted to the registry. The lipase construct was not cloned successfully. Our constructs were designed as follows.
 
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<img src="https://static.igem.org/mediawiki/2017/e/e4/T--Baltimore_Bio-Crew--construct_diagrams.png" style="width: 1000px; height:562px; margin-left:auto;margin-right:auto; display: block;" >  
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<img src="https://static.igem.org/mediawiki/2017/a/a5/T--Baltimore_Bio-Crew--small_construct_diagrams.png" style="width: 1000px; height:562px; margin-left:auto;margin-right:auto; display: block;" >  
 
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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.
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<strong>Lactose Regulated Promoter (BBa_R0011):</strong> a DNA sequence that starts the transcription process in the presence of lactose or IPTG. Thus, the promoter is only active in certain times to regulate the amount of proteins the bacteria is able to produce at a time. The promoter causes the gene to activate and begin the process of forming mRNA and eventually proteins.
 
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Secondly, we had the Ribosome Binding site which signals the start of translation from mRNA to protein.
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<strong>Ribosome Binding Site (BBa_B0030):</strong> an RNA sequence found in mRNA to which ribosomes can bind and initiate translation. It is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation.
 
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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.
+
<strong>PelB Secretion Tag (BBa_K2010002):</strong> allows for bacteria to secrete the enzymes (lipase and esterase) into the periplasm. Once the enzymes are secreted to the periplasm, they are able to exit the cell on their own.
 
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After the PelB tag came the gene for plastic degradation. One construct had chlorogenate esterase, and one construct had lipase.
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<strong>Protein Coding Sequence (BBa_K2187001 for esterase or BBa_K2187000 for lipase):</strong> the DNA sequence is the lipase and esterase gene sequences. The lipase and esterase are both needed to successfully produce the plastic degrading enzymes because both work together to break down and eliminate the plastic particles. One construct contains the chlorogenate esterase coding sequence, and the other construct has the lipase coding sequence.
 
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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.
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<strong>His Tag:</strong> allows us to purify the protein on a nickel column once it’s secreted outside of the bacteria
 
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Finally, our construct contains a terminator that STOPS transcription of the gene sequence.
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<strong>Terminator (BBa_K864600):</strong> a sequence that occurs at the end of the gene and causes transcription to stop
 
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Sponsors
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<h4>
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The Baltimore Bio-Crew thanks our sponsors for their generous support of our team that made our project and travel to the Jamboree possible. Thank you!
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</h4>
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<footer>
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<a href="http://www.bd.com/en-us">
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  <img src="https://upload.wikimedia.org/wikipedia/en/f/f8/Update_Color_BD_PNG_Logo.png" alt="BD Medical Technology, Advancing the World of Health - BD" style="width:100px; height:100px;">
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</a>
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<a href="http://familyleague.org/">
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  <img src="http://baltimoreattendance.org/wp-content/uploads/2015/08/flbcinc-360x230.png" alt="Family League of Baltimore" style="width:100px; height:100px;">
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</a>
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                                      <a>
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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>
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<a href="http://vwrfoundation.org/">
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  <img src="https://static.igem.org/mediawiki/2016/1/1a/T--Baltimore_Biocrew--VWR_Foundation_LOGO.jpeg" alt="VWR Charitable Foundation" style="width:100px; height:100px;">
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</a>
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<a href="http://www.marylandrecyclingnetwork.org/">
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  <img src="https://media.licdn.com/mpr/mpr/shrink_200_200/AAEAAQAAAAAAAAI8AAAAJDY0ZDg0ZjlkLWVlMTItNGI1Mi1iNWEwLWYzMDVlYWMwMTZhZg.png" alt="Maryland Recycling Network" style="width:100px; height:100px;">
 +
</a>
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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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</a>
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</footer>
 
</section>
 
</section>
 
       </body>
 
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</html>

Latest revision as of 19:44, 19 November 2017


BUGSS Logo
Home Team Project Results Design Notebook Composite Part Safety HP Silver Experiment Attribution Collaborations Interlab Contributions

Baltimore Bio-Crew

BALTIMORE BIO-CREW

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

Composite Parts

We designed two constructs, known as biobricks, to allow the bacteria to express the proteins lipase and esterase that will degrade PET plastics. These constructs are identical except for the sequence for the enzyme each construct would produce: esterase and lipase. The chlorogenate esterase construct (BBa_K2436001) was cloned successfully and submitted to the registry. The lipase construct was not cloned successfully. Our constructs were designed as follows.
Lactose Regulated Promoter (BBa_R0011): a DNA sequence that starts the transcription process in the presence of lactose or IPTG. Thus, the promoter is only active in certain times to regulate the amount of proteins the bacteria is able to produce at a time. The promoter causes the gene to activate and begin the process of forming mRNA and eventually proteins.
Ribosome Binding Site (BBa_B0030): an RNA sequence found in mRNA to which ribosomes can bind and initiate translation. It is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation.
PelB Secretion Tag (BBa_K2010002): allows for bacteria to secrete the enzymes (lipase and esterase) into the periplasm. Once the enzymes are secreted to the periplasm, they are able to exit the cell on their own.
Protein Coding Sequence (BBa_K2187001 for esterase or BBa_K2187000 for lipase): the DNA sequence is the lipase and esterase gene sequences. The lipase and esterase are both needed to successfully produce the plastic degrading enzymes because both work together to break down and eliminate the plastic particles. One construct contains the chlorogenate esterase coding sequence, and the other construct has the lipase coding sequence.
His Tag: allows us to purify the protein on a nickel column once it’s secreted outside of the bacteria
Terminator (BBa_K864600): a sequence that occurs at the end of the gene and causes transcription to stop