Difference between revisions of "Team:Austin UTexas/Parts"

 
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<h2>Registry Parts</h2>
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<h2 style="font-family: verdana">Registry Parts</h2>
 
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<h3>Composite Parts</h3>
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<h3 style="font-family: verdana">Basic Parts</h3>
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<p> We submitted two composite PhytoBrick parts to the registry. Each part is described below.  The link to each page is provided in bold text.</p>
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<p style="font-family: verdana"> <b>We submitted two basic PhytoBrick parts and one basic BioBrick part to the registry.</b> Each part is described below.</p>
  
 
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<h6>Constitutive P8 and P32 Lactococcus lactis promoter and RBS composite parts</h6>
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<h6 style="font-family: verdana">Constitutive P8 and P32 <i>Lactococcus lactis</i> promoter and RBS basic parts</h6>
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<p> The P8 and P32 constitutive promoters and RBS sequences are natively found <i>Lactococcus lactis</i>. Although the transcriptional efficiency of these promoters has been characterized and tested in <i>Lactococcus lactis</i> and other Gram-positive bacteria, their functionality in Gram-negative species such as <i>E. coli</i> has not been recorded in the literature. Thus, we assessed the functionality of these promoters in E. coli using the E-2 Crimson reporter gene, which encodes a red fluorescent protein. We have confirmed that <i>E. coli</i> transformed with cassette plasmids containing these P8 and P32 reporter gene constructs are able to successfully express the E2-Crimson red fluorescent protein. As a PhytoBrick compatible part containing the proper overhangs, the P8 promoter can be assembled into a transcriptional unit via Golden Gate assembly method to upregulate expression of a gene of interest in <i>E. coli</i> as well as <i>L. lactis</i>.  
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<p style="font-family: verdana"> The P8 and P32 constitutive promoters and RBS sequences are natively found <i>Lactococcus lactis</i>. These promoters were central to our project, as they were used to direct overexpression of the <i>gadB</i> gene. Although the transcriptional efficiency of these promoters has been characterized and tested in <i>Lactococcus lactis</i> and other Gram-positive bacteria such as <i>Lactobacillus</i>, their functionality in Gram-negative species such as <i>E. coli</i> has not been recorded in the literature. Since we wanted to use <i>E. coli</i> as a chassis for creating our various Golden Gate plasmids, we sought to characterize the functionality of these promoters in <i>E. coli</i> through the use of the <i>E2-Crimson</i> reporter gene, which encodes a red fluorescent protein. We have confirmed that <i>E. coli</i> transformed with cassette plasmids containing these P8 and P32 reporter gene constructs are able to successfully express the <i>E2-Crimson</i> red fluorescent protein. As PhytoBrick compatible parts containing the proper overhangs, the P8 and P32 promoters can be assembled into transcriptional units via the Golden Gate assembly method to upregulate expression of genes of interest in <i>E. coli</i> as well as <i>L. lactis</i> and <i>Lactobacillus</i> species. These PhytoBrick promoter parts have been sequence verified.  
  
 
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The constitutive P8 promoter and RBS PhytoBrick composite part can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2253000">BBa_K2253000</a></b>. The constitutive P32 promoter and RBS PhytoBrick composite part can be found as: <a href="http://parts.igem.org/Part:BBa_K2253001">BBa_K2253001</a></b>.  
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The constitutive P8 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2253000">BBa_K2253000</a></b>. The constitutive P32 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2253001">BBa_K2253001</a></b>.  
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
 
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<p>Remember that the goal of proper part documentation is to describe and define a part so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
 
  
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<h6 style="font-family: verdana">Codon-optimized Blue Chromoprotein basic part</h6>
  
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<p style="font-family: verdana">Chromoproteins are non-fluorescent reporters that indicate a change in a part or device through the appearance of coloration. The blue chromoprotein, <a href="http://parts.igem.org/Part:BBa_K592009">BBa_K592009</a></b>, is genetically unstable due to a high metabolic burden caused by its sequence. Our goal was to attempt to reduce this metabolic load through codon optimization, which favors the use of codons that are in proportions ideal for the specific organism the gene of interest is placed within. Multiple codons can code for the same amino acid but some combinations are used more frequently than others due to an organism’s preference.</p>
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<h5>Note</h5>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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<p style="font-family: verdana">In order to stabilize the blue chromoprotein for long-term use as an effective color reporter, we first ordered a codon-optimized gBlock of the original BBa_K592009 chromoprotein. This sequence was made BioBrick compatible via BioBrick assembly and later inserted into the <a href="http://parts.igem.org/Part:BBa_K608002">BBa_K608002</a></b> vector containing a strong promoter and RBS set within the pSB1C3 backbone. This procedure was done by digesting both the codon-optimized blue chromoprotein and the BBa_K608002 vector, ligating the blue chromoprotein insert to the BBa_K608002 vector, and then conducting a transformation via electroporation. The transformation was then miniprepped and sequence verified.</p>
  
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<p style="font-family: verdana">The codon optimized blue chromoprotein basic part can be found on the iGEM registry as: <a href="http://parts.igem.org/Part:BBa_K2253002">BBa_K2253002</a></b>.
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<h5>Adding parts to the registry</h5>
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<p>The information needed to initially create a part on the Registry is:</p>
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<li>Part Name</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Long Description (Longer description of what the DNA does)</li>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<h5>Inspiration</h5>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h5>Part Table </h5>
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<groupparts>iGEM2016 Example</groupparts>
 
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Latest revision as of 01:14, 2 November 2017

Registry Parts


Basic Parts


We submitted two basic PhytoBrick parts and one basic BioBrick part to the registry. Each part is described below.


Constitutive P8 and P32 Lactococcus lactis promoter and RBS basic parts

The P8 and P32 constitutive promoters and RBS sequences are natively found Lactococcus lactis. These promoters were central to our project, as they were used to direct overexpression of the gadB gene. Although the transcriptional efficiency of these promoters has been characterized and tested in Lactococcus lactis and other Gram-positive bacteria such as Lactobacillus, their functionality in Gram-negative species such as E. coli has not been recorded in the literature. Since we wanted to use E. coli as a chassis for creating our various Golden Gate plasmids, we sought to characterize the functionality of these promoters in E. coli through the use of the E2-Crimson reporter gene, which encodes a red fluorescent protein. We have confirmed that E. coli transformed with cassette plasmids containing these P8 and P32 reporter gene constructs are able to successfully express the E2-Crimson red fluorescent protein. As PhytoBrick compatible parts containing the proper overhangs, the P8 and P32 promoters can be assembled into transcriptional units via the Golden Gate assembly method to upregulate expression of genes of interest in E. coli as well as L. lactis and Lactobacillus species. These PhytoBrick promoter parts have been sequence verified.

The constitutive P8 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: BBa_K2253000. The constitutive P32 promoter and RBS PhytoBrick basic part can be found on the iGEM registry as: BBa_K2253001.

Codon-optimized Blue Chromoprotein basic part

Chromoproteins are non-fluorescent reporters that indicate a change in a part or device through the appearance of coloration. The blue chromoprotein, BBa_K592009, is genetically unstable due to a high metabolic burden caused by its sequence. Our goal was to attempt to reduce this metabolic load through codon optimization, which favors the use of codons that are in proportions ideal for the specific organism the gene of interest is placed within. Multiple codons can code for the same amino acid but some combinations are used more frequently than others due to an organism’s preference.

In order to stabilize the blue chromoprotein for long-term use as an effective color reporter, we first ordered a codon-optimized gBlock of the original BBa_K592009 chromoprotein. This sequence was made BioBrick compatible via BioBrick assembly and later inserted into the BBa_K608002 vector containing a strong promoter and RBS set within the pSB1C3 backbone. This procedure was done by digesting both the codon-optimized blue chromoprotein and the BBa_K608002 vector, ligating the blue chromoprotein insert to the BBa_K608002 vector, and then conducting a transformation via electroporation. The transformation was then miniprepped and sequence verified.

The codon optimized blue chromoprotein basic part can be found on the iGEM registry as: BBa_K2253002.