Difference between revisions of "Team:UST Beijing/Parts"

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                    <li><a href="https://2016.igem.org/Team:UST_Beijing/HP/Gold">Background</a></li>
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                    <li class="divider"></li>
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                  <li><a href="https://2016.igem.org/Team:UST_Beijing/EnzymaticActivity">Enzymatic Activity</a></li>
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                    <li><a href="https://2016.igem.org/Team:UST_Beijing/Description">Recombination</a></li>
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                    <li><a href="https://2016.igem.org/Team:UST_Beijing/Demonstrate">Mixed Fermentation</a></li>
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                    <li><a href="https://2016.igem.org/Team:UST_Beijing/AnimalExperiment">Animal Experiment</a></li>
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<h1 class="intro-lead">Parts</h1>
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<p>We design a new standard BioBrick Part cantral to our project and submit this part to the iGEM registry and we experimentally validate this part works as expected. And more, we document a new application of a BioBrick part from a previous iGEM year. </p>
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</div>
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<h3>Collaborations</h3>
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<li><a href="#part1">New application of BBa_1450004</a></li>
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<li><a href="#part2">Plasmid design</a></li>
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<li><a href="#part3">Plasmid verification</a></li>
  
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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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</ul>
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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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<div id="part1"><h2>New application of BBa_1450004</h2>
  
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<p class="animate-box">The cell wall of notoginseng contains a certain concentration of arabinose, so we expected to use arabinose as Inducer to express T7RNApol. When the concentration of T7RNApol is higher than LacI, T7RNApol would have priority to bind to pET28a plasmid, activate the expression of β-galactosidase. pSB1C3-BBa_1450004 and pET28a-β-galactosidase were transformed into E.coli.  Psb1C3 contains T7 RNA Polymerase gene and can be regulated by pBAD. This double-plasmid system is expected to be regulated by pPAD, and expresses a large number of T7RNA polymerase to inhibit the effect of LacI repression, switch on the expression of β-galactosidase.</p>
  
  
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<img src="https://static.igem.org/mediawiki/igem.org/e/e3/T--UST_Beijing--collaboration02.jpeg" style="width:700px;"></br>
  
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<img src="https://static.igem.org/mediawiki/igem.org/6/60/T--UST_Beijing--collaboration03.jpeg" style="width:700px;"></br>
  
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<p class="animate-box">We added Lac, Ara, PNPG, and bacteria solution in each well of a 96-well palte. After a period of time, we measured the A620 and A450 using microplate reader.( A450 represents the concentration of brokendown PNPG, A620 represents the concentration of bacteria.) After the experiments, we found out that actual A450 could be calculated by this formula：A450real=A450-1.5A620</p>
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<div class="highlight">
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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 class="animate-box">The catalyst efficiency of enzymes expressed by E.coli is represented by A450real/A620</p>
  
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<img src="https://static.igem.org/mediawiki/igem.org/e/e3/T--UST_Beijing--collaboration02.jpeg" style="width:700px;"></br>
  
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<img src="https://static.igem.org/mediawiki/igem.org/6/60/T--UST_Beijing--collaboration03.jpeg" style="width:700px;"></br>
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<p class="animate-box">The result displayed that the concentration of brokendown PNPG decreases when the concentration of lactose increases. Since the the similarities of structure between lactose and PNPG, high concentration lactose has a competitive effect on PNPG. There is an optimum concentration of arabinose as inducer, so less enzymes would be expressed if the concentration of arabinose exceed optimum concentration or is lower than it. The process of bacterial metabolism uses sugar in each well, so that lactose would be less competitive, then enzymes would begin to decompose PNPG.</p>
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<img src="https://static.igem.org/mediawiki/igem.org/6/60/T--UST_Beijing--collaboration03.jpeg" style="width:700px;"></br>
  
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<div class="column half_size">
 
  
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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>
 
 
</div>
 
</div>
  
  
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<div id="part2"><h2>Plasmid design</h2>
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<p class="animate-box">Because double-plasmid system containing ara operon and lac operon may be influenced by complex factors when it works, we connect three gene fragments together on the basis of principle of ara operon. One of the gene fragments can produce araC protein—a type of ara binding protein—inhibiting the activity of pBAD gene. pBAD gene controls the expression of araC gene to one direction and regulates araB, araA, araD genes to another direction. The last gene fragment is a DNA sequence that can expressβ-galactosidase.</p>
  
 +
<p class="animate-box">In this part，we use the orign sequence of araC and pBAD(pC) which copy from genome of E. coli.
 +
Considering the direction of transcription on 5'→3', we replaced the sequences of araB、araA and araD with reverse complementary sequence of β-glucosidase. Terminal J61048 and B0015 are added at the end of coding sequence of araC and β-glucosidase. The prefix and suffix are necessary of parts’ both ends.</p>
  
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<img src="https://static.igem.org/mediawiki/igem.org/f/f8/T--UST_Beijing--collaboration04.jpeg" style="width:700px;"></br>
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</div>
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<div id="part3"><h2>Plasmid verification</h2>
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<p class="animate-box">To validate the availability of plasmid, we test the abilities of decompositing pNPG of three kinds of E.coli strains.</p>
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<img src="https://static.igem.org/mediawiki/igem.org/f/f8/T--UST_Beijing--collaboration04.jpeg" style="width:700px;"></br>
  
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<div class="column half_size">
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<p class="animate-box">We can know that the efficiency of E.coli containing BBa_K2072000 is higher, and the catalyze efficiency of E.coli with adding arabinose is faster than non-arabinose. So it prove that our part can be Induced by arabinose to express β-galactosidase. We speculate that a small amount of enzyme background expression lead the E.coli which is not induced by arabinose to decompose the PNPG.</p>
  
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<h5>What information do I need to start putting my parts on the Registry?</h5>
+
<img src="https://static.igem.org/mediawiki/igem.org/f/f8/T--UST_Beijing--collaboration04.jpeg" style="width:700px;"></br>
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<p>The information needed to initially create a part on the Registry is:</p>
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<ul>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</li>
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<li>Sequence</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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<li>Design considerations</li>
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</ul>
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<p>
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<p class="animate-box">We added E.coli with BBa_K207200, without this part, ONPG and arabinose with varies concentration in different wells. After a period of time, absorbance was measured under 450nm light, the result shew that catalytic effect of arabinose reach peak when its concentration is 7mM, and then it went down while concentration increasing. It was even inhibited when concentration keep increasing.</p>
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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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</div>
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</div>
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</div>
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</div>
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</div>
  
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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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<ul>
 
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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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</ul>
 
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<h5>Part Table </h5>
 
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Revision as of 14:46, 14 October 2016

iGEM team wiki of UST_Beijing

Parts

We design a new standard BioBrick Part cantral to our project and submit this part to the iGEM registry and we experimentally validate this part works as expected. And more, we document a new application of a BioBrick part from a previous iGEM year.

New application of BBa_1450004

The cell wall of notoginseng contains a certain concentration of arabinose, so we expected to use arabinose as Inducer to express T7RNApol. When the concentration of T7RNApol is higher than LacI, T7RNApol would have priority to bind to pET28a plasmid, activate the expression of β-galactosidase. pSB1C3-BBa_1450004 and pET28a-β-galactosidase were transformed into E.coli. Psb1C3 contains T7 RNA Polymerase gene and can be regulated by pBAD. This double-plasmid system is expected to be regulated by pPAD, and expresses a large number of T7RNA polymerase to inhibit the effect of LacI repression, switch on the expression of β-galactosidase.



We added Lac, Ara, PNPG, and bacteria solution in each well of a 96-well palte. After a period of time, we measured the A620 and A450 using microplate reader.( A450 represents the concentration of brokendown PNPG, A620 represents the concentration of bacteria.) After the experiments, we found out that actual A450 could be calculated by this formula：A450real=A450-1.5A620

The catalyst efficiency of enzymes expressed by E.coli is represented by A450real/A620



The result displayed that the concentration of brokendown PNPG decreases when the concentration of lactose increases. Since the the similarities of structure between lactose and PNPG, high concentration lactose has a competitive effect on PNPG. There is an optimum concentration of arabinose as inducer, so less enzymes would be expressed if the concentration of arabinose exceed optimum concentration or is lower than it. The process of bacterial metabolism uses sugar in each well, so that lactose would be less competitive, then enzymes would begin to decompose PNPG.


Plasmid design

Because double-plasmid system containing ara operon and lac operon may be influenced by complex factors when it works, we connect three gene fragments together on the basis of principle of ara operon. One of the gene fragments can produce araC protein—a type of ara binding protein—inhibiting the activity of pBAD gene. pBAD gene controls the expression of araC gene to one direction and regulates araB, araA, araD genes to another direction. The last gene fragment is a DNA sequence that can expressβ-galactosidase.

In this part，we use the orign sequence of araC and pBAD(pC) which copy from genome of E. coli. Considering the direction of transcription on 5'→3', we replaced the sequences of araB、araA and araD with reverse complementary sequence of β-glucosidase. Terminal J61048 and B0015 are added at the end of coding sequence of araC and β-glucosidase. The prefix and suffix are necessary of parts’ both ends.


Plasmid verification

To validate the availability of plasmid, we test the abilities of decompositing pNPG of three kinds of E.coli strains.


We can know that the efficiency of E.coli containing BBa_K2072000 is higher, and the catalyze efficiency of E.coli with adding arabinose is faster than non-arabinose. So it prove that our part can be Induced by arabinose to express β-galactosidase. We speculate that a small amount of enzyme background expression lead the E.coli which is not induced by arabinose to decompose the PNPG.


We added E.coli with BBa_K207200, without this part, ONPG and arabinose with varies concentration in different wells. After a period of time, absorbance was measured under 450nm light, the result shew that catalytic effect of arabinose reach peak when its concentration is 7mM, and then it went down while concentration increasing. It was even inhibited when concentration keep increasing.

北京科技大学