Team:Lambert GA/Project

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Biobricks:
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Biobricks are defined as the “…standard for interchangeable parts, developed with a view to building biological systems in living cells” by the iGEM Parts website. Our contribution to CDA would be essential to biobrick because it will allow others to use the part in the future. The biobrick that is created will be mapped out and put into a database which is basically a free dictionary of biobricks. This compilation of biobricks allow others to use them in their experiments, expand on, or restructure. Without a database of biobricks, we would have a hard time expanding on our knowledge and would be continuously having to try to invent them for ourselves.
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Jaemor Farms:
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On November 25th, the Lambert iGEM team went to Jaemor Farms to interview the local farmers and producers of fresh fruits and vegetables. The main objective was to find out how spoiled produce really affected the agricultural industry, such as in amount of profit lost per year due to spoiled peaches. The team toured the farm, interviewed the farm’s owner, and had a great time eating fresh homemade desserts. We came back with some impressive statistics- in 2013, Jaemor Farms produced 700,350 pounds of peaches, 124,949 pounds of strawberries, 115,204 pounds of tomatoes, and 48,000 pounds of apples, among the over 300 different products produced. However, according to global stats, over 50% of fresh produce is lost to spoilage every year across the world. That translates to only half of all the peaches harvested that will make it to the grocery store. With this percentage in mind, the previous numbers cited now seem very small in retrospect. Hunger is a huge problem in less developed countries, and still plagues 1 out of every 6 person in the US. If the LHS iGEM team’s 2014 project is to succeed, it can potentially save world hunger in the long run by preventing food spoilage and increasing the “shelf-life” of fresh produce, which would then go to mean more food available for food aid or distribution to those that really need it.
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Use of Cells for Chitin
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Chitin, which is mostly commonly found in the cell walls of fungi and the shells of crustaceans, is the second most abundant natural long-chain polymer. Due to the many industrial and medical applications of chitin’s deacetylated form, chitosan, the extraction of chitin from chemical and biological sources remains very important, especially to our experiment.
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Chitin and chitosan are mainly extracted through the demineralization, deproteinization, and other decomposition methods, of crustacean shells and fungi cell wall using acids and bases (mainly hydrochloric acid). The principal problem of this method is that dense acid concentrations cause hydrolysis (the cleavage of chemical bonds due the addition of water) of the polymer, which can potentially change the physical and chemical properties of the outcome, lowering the quality of the final product. This problem leads to more interest in more organic extractions through designer cells.
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Changing the plasmids of designer cells to express chitosan in the presence of chitin and chitin deacetylase allows for higher quality products, which allows more efficient applications and less acid-base dependency.
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<li class="first"><a href="https://2013hs.igem.org/Team:Lambert_GA" accesskey="1" title="">Home</a></li>
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                <li><a href="https://2013hs.igem.org/Team:Lambert_GA/team" accesskey="3" title="">Team</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lambert_GA/project" accesskey="2" title="">Project</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lambert_GA/labnotebook" accesskey="4" title="">Lab Notebook</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lambert_GA/resultsconclusions" accesskey="5" title="">Conclusions</a></li>
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                <li><a href="https://2013hs.igem.org/Team:Lambert_GA/futureaspirations" accesskey="8" title="">Future</a></li>
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<li><a href="https://2013hs.igem.org/Team:Lambert_GA/safety" accesskey="5" title="">Safety</a></li>
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                <li><a href="https://2013hs.igem.org/Team:Lambert_GA/attributions" accesskey="6" title="">Attributions</a></li>
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                <li><a href="https://2013hs.igem.org/Team:Lambert_GA/humanpractices" accesskey="7" title="">Human Practices</a></li>           
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                <li><a href="https://2013hs.igem.org/Team:Lambert_GA/activities" accesskey="6" title="">Activities</a></li>             
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<font size=4><center><b><u><h1>Project</h1></u></b></center></font>
 
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<center><img src="https://fbcdn-sphotos-a-a.akamaihd.net/hphotos-ak-prn1/p526x296/1016042_213819975433663_2086842284_n.jpg" width="450"/></center>
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How to get Chitosan Out of Cells
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<font size=3><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In 2010, Georgia Institute of Technology’s iGem team submitted the part K410000, a periplasmic heat generator made with HybB and OmpA and AOX. The following year several teams used HybB in their projects with mixed results.  Our project is built off of these projects  and it's purpose is to characterize and Biobrick HybB, a cold shock promoter.A Biobrick is a sequence of DNA that has standard prefixes and suffixes. Our goal is to test the reliability of HypB, the cold shock promoter, in order to use temperature to regulate protein expression. Our test will be to put RFP under HybB promotion and give it a cold shock treatment, which should enable HybB to act as a switch and turn on the red fluorescent protein in the <i> E. Coli</i> cells, if the HybB cells are reliable. We have decided to use HybB, even though it yeilded mix results in the past, because this cold shock promoter showed potential.<br><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HybB was synthesized for the first time by the 2005 UCSF iGEM team. The 2006 MIT iGEM team standardized HybB and submitted it to the parts registry. HybB has the part number J45503 and is not available from the Parts Registry. Georgia Tech’s 2010 iGEM team engineered a chassis to include HybB, OmpA, and AOX. The 2011 Leuven iGEM team reported, “We see that promoter activity is induced when cells are transferred to 25°C and even when they are put in an ice bath (4°C). Unfortunately, however, cells that are kept at 37°C also display an increase in promoter activity, indicating leakiness in the system.” The 2011 Groningen team reported that the HybB Promoter did not work. Through sequencing, they proved that HybB was found in the cells, and they ran the reactions at the same temperatures.However, they could not get the same results as the Leuven team. Due to these more recent findings, we are not sure whether HybB works or not.<br><br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We obtained HybB from Georgia Tech's 2010 iGEM Team, but upon sequencing and experimentation, we learned that the stock was not reliable.  We then designed primers to isolate the proposed HybB promoter sequence from the <i>E. Coli</i> DH10 genome. </font>
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Chitosan is a vital part of this project. Chitosan is produced from raw materials that naturally contain chitin. Chitosan’s biological sources are often crustacean shells, specifically shrimp shells, and its assay is greater than or equal to 75 percent (deacetylated). The manufacturing process of shrimp leaves behind a waste shell. This shell is then crushed to remove about 90 percent of the protein present. The crushed shells are washed with salt water and decalcified with a diluted form of hydrochloric acid. The shells are washed once again with recycled salt water and deproteinized with diluted sodium hydroxide. The treated shell produces chitin at this point. The chitin is put through deacetylation and treated with concentrated sodium hydroxide to produce chitosan.
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Revision as of 15:07, 14 June 2014

Biobricks:

Biobricks are defined as the “…standard for interchangeable parts, developed with a view to building biological systems in living cells” by the iGEM Parts website. Our contribution to CDA would be essential to biobrick because it will allow others to use the part in the future. The biobrick that is created will be mapped out and put into a database which is basically a free dictionary of biobricks. This compilation of biobricks allow others to use them in their experiments, expand on, or restructure. Without a database of biobricks, we would have a hard time expanding on our knowledge and would be continuously having to try to invent them for ourselves.

Jaemor Farms:

On November 25th, the Lambert iGEM team went to Jaemor Farms to interview the local farmers and producers of fresh fruits and vegetables. The main objective was to find out how spoiled produce really affected the agricultural industry, such as in amount of profit lost per year due to spoiled peaches. The team toured the farm, interviewed the farm’s owner, and had a great time eating fresh homemade desserts. We came back with some impressive statistics- in 2013, Jaemor Farms produced 700,350 pounds of peaches, 124,949 pounds of strawberries, 115,204 pounds of tomatoes, and 48,000 pounds of apples, among the over 300 different products produced. However, according to global stats, over 50% of fresh produce is lost to spoilage every year across the world. That translates to only half of all the peaches harvested that will make it to the grocery store. With this percentage in mind, the previous numbers cited now seem very small in retrospect. Hunger is a huge problem in less developed countries, and still plagues 1 out of every 6 person in the US. If the LHS iGEM team’s 2014 project is to succeed, it can potentially save world hunger in the long run by preventing food spoilage and increasing the “shelf-life” of fresh produce, which would then go to mean more food available for food aid or distribution to those that really need it.

Use of Cells for Chitin

Chitin, which is mostly commonly found in the cell walls of fungi and the shells of crustaceans, is the second most abundant natural long-chain polymer. Due to the many industrial and medical applications of chitin’s deacetylated form, chitosan, the extraction of chitin from chemical and biological sources remains very important, especially to our experiment. Chitin and chitosan are mainly extracted through the demineralization, deproteinization, and other decomposition methods, of crustacean shells and fungi cell wall using acids and bases (mainly hydrochloric acid). The principal problem of this method is that dense acid concentrations cause hydrolysis (the cleavage of chemical bonds due the addition of water) of the polymer, which can potentially change the physical and chemical properties of the outcome, lowering the quality of the final product. This problem leads to more interest in more organic extractions through designer cells. Changing the plasmids of designer cells to express chitosan in the presence of chitin and chitin deacetylase allows for higher quality products, which allows more efficient applications and less acid-base dependency.


How to get Chitosan Out of Cells

Chitosan is a vital part of this project. Chitosan is produced from raw materials that naturally contain chitin. Chitosan’s biological sources are often crustacean shells, specifically shrimp shells, and its assay is greater than or equal to 75 percent (deacetylated). The manufacturing process of shrimp leaves behind a waste shell. This shell is then crushed to remove about 90 percent of the protein present. The crushed shells are washed with salt water and decalcified with a diluted form of hydrochloric acid. The shells are washed once again with recycled salt water and deproteinized with diluted sodium hydroxide. The treated shell produces chitin at this point. The chitin is put through deacetylation and treated with concentrated sodium hydroxide to produce chitosan.

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