Team:Tsinghua/project/outline

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<div id="main_content"><a name="outline"></a>
<div id="main_content"><a name="outline"></a>
<h1>Outline</h1>
<h1>Outline</h1>
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<a name="mod1"></a></html>
 
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=='''Comparison between Antibody Recombination and Our Recombination System'''==
 
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<html><div class="content_block"></html>
 
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Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens.
 
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Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.
 
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It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.
 
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<div class="content_block">
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==Module I==
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<br/>
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In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.
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<br/>
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[[Image:TSModule1.PNG|650px]]
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<table border="2" bordercolor="maroon" bgcolor="silver">
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<tbody>
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<tr>
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<th colspan=3>Antibody Production</th>
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<th width=50px></th>
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<th colspan=2>E Coli. Production System</th></tr>
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<tr>
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  <td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td>
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  <td rowspan=3 width=70px>Preparation</td>
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  <td width=70px>RSS Sequence</td>
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  <td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td>
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  <td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td>
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  <td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td>
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</tr>
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<tr>
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  <td>VDJ Recombinase</td>
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  <td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td>
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  <td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td>
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</tr>
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<tr>
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  <td>VDJ Library</td>
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  <td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td>
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  <td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td>
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</tr>
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<tr>
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  <td>Recombination</td>
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  <td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td>
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  <td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td>
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</tr>
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<tr>
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  <td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td>
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  <td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td>
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  <!---------<td>Junctional Diversity</td>---------------------->
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</tr>
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<tr>
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  <td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td>
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  <td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td>
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</tr>
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<tr>
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  <td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td>
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  <td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td>
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</tr>
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<tr>
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  <td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td>
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</tr>
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<tr>
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  <td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td>
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</tr>
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</tbody>
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</table>
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</html>
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<br/>
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Generally speaking, antibody production in our project can be divided into two modules.
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===Abstract===
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<body bgcolor="#336699" text="#ffffff" link="#60a179">
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<br/>
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<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span>
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<br/><br/>
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<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span>
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<br/><br/>
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</html>
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===Flow chart===
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==Project Design==
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[[Image:THUProjectFlowChart.JPG|600px]]
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===Landing Pad Construction and Insertion===
 
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<br>
 
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'''Purpose of this step'''
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===Module 1===
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Landing pad insertion is the first step of our two-step recombination system.
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'''Antibodies Library Diversity & Randomicity'''
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We insert a “landing pad” fragment which includes a promoter (placIQ1) and a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 20-bp landing pad regions (LP1 and LP2) into Escherichia coli chromosome via att recombination. Then the helper plasmids encoding I-SceI endonuclease and λ-Red and donor plasmid encoding various antibiotic genes flanked by I-SceI recognition sites and same landing pad regions (LP1 and LP2) are transformed, which will be introduced in detail in next part. I-SceI expression is induced via the addition of L-arabinose. I-SceI recognition sites in the donor plasmid and chromosome are cleaved. Integration of the fragment in donor plasmid is facilitated by IPTG-induced λ-Red expression.
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[[Image:TSModule1.PNG|650px]]
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This two-step recombination method allows for the insertion of very large fragments into a specific location in ''Escherichia coli'' chromosome and in any orientation.
 
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The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.
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'''Construction of landing pad'''
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Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination, the manner in which lambda phage integrates its DNA into E Coli genome. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):
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Landing pad consists of the following parts: a promoter (placIQ1), two landing pad regions, two I-SceI recognition sites and a tetracycline resistance gene (tetA).
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1)25bp-long random sequence
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[[Image:THUProjectFigure1.PNG|650px]]
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2)15bp-long recognition sequence of restriction enzyme I-scel
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Another construction strategy is shown as below:
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3)  antibiotic resistance gene used for antibody selection
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[[Image:THUProjectFigure1_1.JPG|650px]]
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4)  15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)
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'''The first construction is used as the four-gene donor plasmid method, while the latter is for the two-gene donor plasmid method.''' We take the advantage of the former construction in the final recombination, because the promoter will not be cut when the recombination happens, thus saving us lots of time. As the latter construction, we can easily add a promoter before the gene, in two-gene donor plasmid A(shown in the Donor Plasmid Construction Part).
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5)  25bp-long random sequence (corresponding to 1)
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First we obtain two target DNAs – promoter and tetA with PCR. Then We use the overlap extension polymerase chain reaction (or OE-PCR) to link two fragments together. Primer 2 and primer 3 have homologous sequences, so one segment can anneal to the other in certain conditions, in other words, the two segments can overlap into one segment after extension. If necessary, operate PCR again with primer 1 and primer 4 to obtain more products.  
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After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination.  
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<br>
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Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.
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'''ATT RECOMBINATION'''
 
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Our approach is based on genome targeting systems that utilize plasmids carrying a conditional-replication origin and a phage attachment (attP) site (17). We refer to our plasmids as CRIM (conditionalreplication, integration, and modular) plasmids. CRIM plasmids can be integrated into or retrieved from their bacterial attachment (attB) site by supplying phage integrase (Int) without or with excisionase (Xis) in trans.
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design.  
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We got a strain with plasmid pUK2 from LAB. Than we develop a E.coli strain contains the helper plasmid AH69. These two plasmids are shown below. In order to match other parts of our whole project, the modification that kan-exon should be replaced with tet-SDS was necessary. We got tet-SDS from another plasmid named pkts-cs. Then we use PCR to get the two fragments as PT (from pkts-cs) and V (from pUK2). We ligate PT and V after digestion and modification to form a new plasmid. This new plasmid was named as pUKIP and contains a promoter region, a tet-SDS region and phage attachment site (attP). As reported in the paper, there exist one bacterial attachment site (attB) of HK002 in TorT-TorS gene of the chromosomal DNAs of E.coli K12 strain. That is to say, the PT fragment will integrate into the cell genome after the plasmid was transferred in cells.
 
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[[Image:THUProjectFlowchart.jpg|700px]]
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PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.
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===Helper Plasmid(HP) Insertion===
 
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'''CRIM plasmid integration'''
 
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Cells carrying a CRIM helper plasmid were grown in 20 ml of LB cultures with ampicillin at 30°C to an optical density of 600 nm of ca. 0.6 and then made electrocompetent. Following electroporation, cells were suspended in LB without ampicillin, incubated at 30°C for 30 min, at 42°C for 30 min and at 30°C for 30 min, and then spread onto selective agar (tet) and incubated at 37°C. Colonies were purified once nonselectively and then tested for antibiotic resistance for stable integration and loss of the helper plasmid and by PCR for copy number.
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After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination.  
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'''CRIM plasmid excision'''
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In this plasmid(Helper Plasmid), there are two genes used for recombination.
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Cells were transformed with the respective Xis/Int CRIM helper plasmid and then spread on ampicillin agar media at 30°C. Colonies were purified once or twice nonselectively on plates that were incubated for 1 h at 42°C and overnight at 37°C. They were then tested for antibiotic sensitivities and by PCR for loss of the integrated plasmid.
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The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.
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<br>
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===Donor Plasmid(DP) Construction===
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The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.
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'''Purpose of this step:'''
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After inserting landing pad and helper plasmid to E.coli, we must construct a series of donor plasmids to demonstrate that this system can truly realize the recombinant process in E.coli, thus we can further use this module to simulate the recombination of antibody gene in mammalian B cells. We not only need to test the efficiency of recombination, but also ensure that genes we get from this recombinant process can be expressed correctly and have their original function. So we intend to construct three plasmids to test the system.
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In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.
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'''Experiment design and expected results:'''
 
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[1] Donor plasmid A
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.
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[[Image:THUProjectFigure2.jpg|650px]]
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In donor plasmid A, we insert two genes, kanamycin resistant gene (Kanr) and Chloromycetin resistant gene (Chlr). At the 5’ end of these two genes, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.
 
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After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down these two genes (containing recombination sequences), which can recombine with the bacterial chromosome. In our expectation, either kanr or chlr will replace the landing pad, resulting in the bacteria resistance to either kanamycin or chloromycetin, but not both. This process will be random, so we can get as many colonies resistant to kanamycin as those resistant to chloromycetin.
 
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[2] Donor plasmid B
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Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:
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[[Image:THUProjectFigure3.jpg|650px]]
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Donor plasmid B includes four genes, GFP, mCherry, Kanr, Chlr, respectively. The same with genes in donor plasmid A, we add recombination sequences and I-scel recognition sequences to the ends of each genes. The recombination sequences of GFP and mCherry are identical, and those of Kanr and Chlr are the same. Note that recombination sequence at 3’ end of GFP (mCherry) and that at 5’ end of Kanr (Chlr) are the same, so we can get a random recombination of two genes, one is a fluorescence gene and the other is resistant gene, creating 2X2=4 different results.
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1) one 15bp-long restriction enzyme I-scel recognition site
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[3] Donor plasmid C
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2) one 25bp-long random sequence
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[[Image:THUProjectFigure4.JPG|650px]]
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For constructing donor plasmid C, we first cut the GFP and mCherry to 2 fragments respectively. Then we insert these four fragments into the plasmid in the order shown in the above picture. We expect that we can see either green or red fluorescence after transforming and inducing. Through this experiment, we can tell whether or not recombination sequence will affect the normal function of genes, further demonstrate that antibody producing by our system will be effective. On the other hand, we should note that the sequence length is another significant reason of antibody diversity, so the effect of recombination sequence on antibody will much small than that on fluorescence genes.
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3) one fragment for insertion and recombination
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====Strategies====
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4) one 25bp-long random sequence
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Traditionally, we can construct these three plasmids by inserting genes into the plasmid one by one. However, considering the huge number of antibody fragments, we try our best to seek other strategies to complete the ligation of multiple fragments, which can be done more quickly and efficiently.
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5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)
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=====Principle=====
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(Note: the order of the five parts differ from that of landing pad)
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The fragments include different landing pad regions and endoclease recognition site, and are grouped together by sharing the same landing pad region.
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As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.
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=====Method=====
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In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.
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In the demand of larger amount of fragments and constructed plasmids, we find two ways to realize such purpose, regarded as the key point of our whole project-guarantee the large variation of antibody.
 
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[[Image:THUProjectFigure5.JPG|650px]]
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design.  
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[[Image:THUProjectFigure6.JPG|650px]]
 
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===Removal of Helper Plasmid(HP)===
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As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.
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Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.
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===Donor Plasmid(DP) Insertion & Recombination Induction===
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In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.
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'''Propose of this step:'''
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After the reform of the E.coli genome and the construction of the donor plasmid, we need to test our module’s function. First we should insert the donor plasmid(DP) into the E.coli, and then induce the recombination by adding IPTG and arabinose. Arabinose active the I-Sel restriction enzyme, then cut DP and genome at the same site. After the digestion, we add IPTG to help the recombination, using the homologous sequence near the cohesive end.
 
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'''DP Insertion: '''
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction.
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As we said before, we use a pre-altered template to amplify landing pad fragments using the landing pad regions as standardized priming sites. Here we used the conventional electroporation method to transform the DP into E.coli genome, then incubate the plate at 37°overnight.
 
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Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is a dynamic phenomenon that depends on the local transmembrane voltage (we used 1 V) at each point on the cell membrane. If E.coli and DP are mixed together, the plasmids can be transferred into the cell after electroporation. This procedure is highly efficient than chemical transformation. (Partly from wikipedia)
 
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[[Image:THUProjectFigure7.png|650px]]
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Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.
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'''Recombination Induction:'''
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Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.
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Individual colonies were inoculated into 5 ml of EZ-Rich Defined Medium (RDM; Teknova) +0.5% glycerol, 2mM IPTG, and 0.2% w/v L-arabinose. After growing at 37°C for 1 h in a shaking water bath, we transfer the medium to 30_C shaking water bath for 4 h, then 100 mg/ml spectinomycin was added. At first the I-Sel enzyme is expressed to cut the genome and DP at the same site. And this step is used to constitutive express the Rec A enzyme, thus initiating the recombination by the homologous region.
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Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.
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The appropriate antibiotic for the given insertion fragment was then added (25 mg/ml kanamycin, 34 mg/ml chloramphenicol), and the cultures were grown overnight. The next day, 100 ml sample was plated on LB plates with the appropriate antibiotic and grown at 37°C. We test the sample by screening it on LB plates containing 100 mg/ml ampicillin or 10 mg/ml tetracycline to verify the loss of the landing pad and donor plasmid.
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[[Image:THUProjectFigure8.JPG|650px]]
 
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<html></div><a name="mod2"></a></html>
 
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==Module II==
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part.
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<html><div class="content_block"></html>
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===Strategy 1===
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Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.
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'''Bacterial Based Microarray'''
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[[Image:TSModule2m.PNG|500px]]
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate.  
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====Outline of screening desired antibody by bacterial based microarray====
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Microarray technique is a kind of pretty mature biomedical technology applied to diverse areas from pharmacology research to clinical use, such as genetics diagnose. Due to the advantage that large amount of various substances can be screened in microarray, microarray can be used for selection of desired antibodies with specific affinity in our project. However, current existing microarray technology rely on specific DNA or protein attached substrate and our project lean on bacteria expressing specific surface protein used for screening for desired antibody. Therefore, traditional protocols and materials used in microarray will be adjusted in our project. We believe that our adjustment will improve the efficiency of antibody screening and help our project achieve its ultimate goal.
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====Specific description of the details of the experiments====
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<br/><br/>
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In this part, we want to transform two batches of E coli with vectors carrying specific genes expressing various kinds of antibody achieved by the recombination methods in our project and specific antigen or some other protein used to select antibody from the aforementioned mix of antibodies. By fusion antibody and antigen gene with the genes of membrane integral displaying protein called OmpA, we can manage to display our protein to the surface of the bacteria. Therefore, the selection will process through interaction between antibody and antigen at the surface of the bacteria. Then, by linking the gene of display protein to the gene of one kind of protein called cellulose binding domain which can bind to cellulose, we are able to anchor the bacteria expressing CBD to the surface of the specific microarray coated with cellulose substrate. The whole process is illustrated in the following figure.
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[[Image:THUProjectFigure9.jpg|650px]]
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====Information concerning the specific proteins====
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===Module 2===
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=====Cellulose binding domain=====
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The protein used in this part comes from CipC gene found in Clostridium cellulolyticum. CipC gene encodes a protein called cohesion which participate in degradation of cellulose for the benefits of the bacteria Clostridium cellulolyticum. The protein encoded in this gene contains a specific domain called cellulose binding domain which bind to cellulose with pretty high binding affinity and thus facilitate the degradation of the cellulose. The structure of CBD has been resolved shown in the figure below. Our purpose is to utilize the ability of CBD to bind to cellulose and express CBD in E coli and thus force it to bind to microarray coated with cellulose substrate.
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[[Image:THUProjectFigure10.jpg|200px]]
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The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.
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=====Displaying protein OmpA=====
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The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.
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At present, two kinds of membrane protein are known, that is, alpha helix protein from cytoplasmic membrane and beta-barrel protein in bacteria outer-membrane, exemplified by porin protein. There are two membrane layers in gram-negative bacteria, separately called inner membrane and outer membrane. OmpA is located in outer membrane and belongs to beta-barrel category. This protein is responsible for normal physiological functions in gene regulation in E coli. Because OmpA is located in outer membrane, intensive investigation has been conducted in OmpA because OmpA plays an important role in signal transduction and much work has focused on the mechanism how OmpA was transported from cytosol out of the membrane. Because of the clarified function of the protein, some bioengineers managed to utilize its function to display protein to achieve certain purposes. Therefore, we try to take advantage of the properties of OmpA to display protein, that is, to display CBD, antibody and antigen. The structure of OmpA has been determined, illustrated in the following figure.
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In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.
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[[Image:THUProjectFigure11.jpg|300px]][[Image:THUProjectFigure12.jpg|300px]]
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Previous work has demonstrated that 46-159 amino acid is adequate to anchor OmpA to membrane and from the topology figure, we can find out that the fragment of AA 46-159 comprise the transmembrane part of the protein, as the red box indicates. Besides, to ensure the transport of OmpA from cytosol to cell membrane, we need to add a signal peptide to the N terminal of OmpA. Previous work found that one kind of lipoprotein in E coli contains a signal peptide consisting of nine amino acids, which ensures the successful transport of the protein outside the membrane. Mining through the parts provided by iGEM, we found one part which provides exactly the same sequence we wants. This part contains N terminal signal peptide and the sequence AA 46-159 and the linker region downstream of OmpA which ensures the folding of the attached domain, such as CBD. The engineered OmpA is illustrated as following.
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To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.
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[[Image:THUProjectFigure13.jpg|500px]]
 
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=====The selection of antibodies=====
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.
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Due to commercialization of antibody, we have no access to the cDNA of these antibodies. Therefore, we managed to synthesize the cDNA.
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=====Design of microarray=====
 
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Microarray is a solid substrate based 2 dimensinoal array, which can be utilized to detect large sum of bio-substance. After decade of developing, various kinds of microarrays have been invented, such as DNA microarray, protein microarray, tissue microarray and so on. Different microarrays can be used to detect different components based on different substrates. Our project belongs to cellular microarray, aiming to select different kinds of antibodies based on the interaction between different proteins displayed on bacteria membrane.
 
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This part of our project aims to attach CBD-expressing bacteria to the surface of microarray coated with cellulose. Weizmannn Institute has developed zephyrin-based microarray, in which the microarray plate is dotted with cellulose. Thus, this innovation provides potential for application in our project.
 
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===Strategy 2===
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In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.
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'''Transmembrane Signaling Pathway Method'''
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[[Image:TSModule2s.PNG|500px]]
 
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===Outline===
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PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.
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In order to select a specific antibody from a large number of antibodies, we studied the formation of mammalian antibodies. In the process of antibody production in mammals, mediated Annexin antibodies-mediated antibody activation play an important role. This section hopes to use a membrane receptor of E. coli to simulate this process, to achieve
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the purpose of screening.
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To achieve this, a ToxR-based two-hybrid system was introduced into E.coli cells. The Vibrio cholerae transcriptional regulator ToxR is anchored in the cytoplasmic membrane by a single transmembrane segment, its C-terminal domain facing the periplasm. Most of its N-terminal cytoplasmic domain shares sequence similarity with the winged helix–turn–helix (wHTH) motif of OmpR-like transcriptional regulators. The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera.
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In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.
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ToxR stimulates transcription from the cholera toxin gene promoter ctx by direct binding to DNA element ITITTGAT present in different isolates of Vcholerae in three to eight tandemly repeated copies upstream of the ctxAB structural genes.
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Transcription activation is thought to be initiated by environmental stimuli which cause the periplasmic ToxR domain to form a homodimer. This, in turn, tethers together the two cytoplasmic ToxR domains, which can now bind to the control region of the ctx promoter. A second membraneassociated protein, ToxS (Mr 19 000), is required for maximal activation of the ctx promoter; most plausibly it stabilizes the ToxR dimer by direct contact.
 
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[[Image:THUProjectFigure14.jpg|600px]]
 
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Figure . Original structure of ToxR system in Vibrio cholerae
 
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Fusion protein was made between the inner- and trans-membrane parts of ToxR and of the recombinant antibody.
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PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.
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[[Image:THUProjectFigure15.jpg|100px]]
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When the specific antigens recognized by antibodies, antibodies with the same antigen recognize site will be close to each other through the antigen,inner-membrane part of “Antibody-ToxR” Protein dimerize, ctx promoter active, Resistance gene starts to be transcribed
 
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[[Image:THUProjectFigure16.jpg|600px]]
 
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Use “Anti-Histag Antibody” and “Protein with His-tag” as “antibody” and “antigen” to verify the effect of this system.
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Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens.  
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Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.
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Antibody: ToxR + Anti-Histag Antibody scFv Light Chain
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It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.
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Plus:    ToxR + Anti-Histag Antibody scFv Heavy Chain
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Antigen: His tag + Protein A + Histag
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Protocol
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We need to achieve the construction of the selection vector under the control of ctx promoter, then induce to the bacteria.
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Measure the expression of the two genes and measure the distribution in membrane.
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Use the exogenous protein carried His-tag as the antigen.
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Treat with antibiotics.
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Compare the expression level between the experiment group and the control, then define the effect of our system indirectly.
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Reference:A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein–protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation
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Reference:A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein–protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation
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===Strategy 3===
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Cooperation with <html><a href="https://2010.igem.org/Team:Macquarie_Australia" target=blank>Macquarie Australia</a>
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</div>
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Latest revision as of 17:16, 27 October 2010

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Outline


In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.
Antibody Production E Coli. Production System
VDJ Recombination Preparation RSS Sequence Landing Pad Insertion Module I: Recombination System
VDJ Recombinase Helper Plasmid Insertion
VDJ Library Donor Plasmid Construction
Recombination DP Insertion and Recombination Induction
Somatic Hypermutation
CBD-based Microarray Module II: Selection System
Antigen Selection
ToxR-based Transmembrane Pathway
Cooperation With Macquarie_Australia

Generally speaking, antibody production in our project can be divided into two modules.


Module I: Generation of antibody library

Module II: Selection of specific antibodies

Project Design

Module 1

Antibodies Library Diversity & Randomicity TSModule1.PNG


The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.

Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination, the manner in which lambda phage integrates its DNA into E Coli genome. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):

1)25bp-long random sequence

2)15bp-long recognition sequence of restriction enzyme I-scel

3) antibiotic resistance gene used for antibody selection

4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)

5) 25bp-long random sequence (corresponding to 1)

After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination.

Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.


PLEASE refer to 'Landing Pad Construction and Insertion' to learn about the details of Landing Pad design.


PLEASE refer to this 'link' to learn more about ATT recombination.


After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination.

In this plasmid(Helper Plasmid), there are two genes used for recombination.

The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.

The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.

In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.


PLEASE refer to 'HP insertion' to learn more about Helper Plasmid.


Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:

1) one 15bp-long restriction enzyme I-scel recognition site

2) one 25bp-long random sequence

3) one fragment for insertion and recombination

4) one 25bp-long random sequence

5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)

(Note: the order of the five parts differ from that of landing pad)

As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.

In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.


PLEASE refer to ‘DP construction’ to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design.


As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.

In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.


PLEASE refer to 'DP construction and Strategies' for the methods of rapid plasmid construction.


Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.

Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.

Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.


PLEASE refer to ‘DP Insertion and Recombination Induction’ to learn more about this part.


Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.


PLEASE refer to ‘Result’ to learn more about the identification of recombination rate.



Module 2

The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.

The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection. In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.

To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.


PLEASE refer to ‘ToxR-based Transmembrane Signaling Pathway Method’ for detailed description of this method.


In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.


PLEASE refer to ’Bacterial based microarray’ for details.


In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.


PLEASE refer to this 'link' to learn more about our cooperation.


Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. Our system is aiming at imitating this recombination process, using E.coli as the gene carrier. It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.