Difference between revisions of "Team:Georgia State/Composite Part"

 
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     <title>Composite Part</title>
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<div class="container"
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<section class="bg-gray section-padding-80">
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<p>Dino III GFP Plasmid<p>
+
<div class="container">
−
<p><li><a href="http://parts.igem.org/Part:BBa_K3042005">Part:BBa_K3042005</a></li><p>
+
 
−
<p>Dino III RFP Plasmid<p>
+
<div class="row center mt-80 mb-80">
−
<p><li><a href="http://parts.igem.org/Part:BBa_K3042006">Part:BBa_K3042006</a></li><p>
+
<div class="table responsive">
−
<p>pCB302-gfp-MBD plasmid<p>
+
<table class="table-striped table-bordered w-auto overflow-auto" height="500" width="500">
−
<p>*This part is not in the registry*<p>
+
<tr align="align">
 +
<th colspan="2"><h4>Composite Parts</h4></th>
 +
</tr>
 +
<td><h6>Dino III GFP Plasmid</h6></td>
 +
<td><li><a href="http://parts.igem.org/Part:BBa_K3042005">Part:BBa_K3042005</a></li></td>
 +
</tr>
 +
<tr><td><h6>Dino III RFP Plasmid</h6></td>
 +
<td><li><a href="http://parts.igem.org/Part:BBa_K3042006">Part:BBa_K3042006</a></li></td>
 +
</tr>
 +
<tr><td><h6>pCB302-gfp-MBD plasmid</h6></td>
 +
<td><h6>*This part is not in the registry*</h6></td>
 +
</table>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
<h1>pCB302-gfp-MBD Plasmid</h1>
 +
<div class="row">
 +
<div class="col-6">
 
<p>Our predicted sequence created in SnapGene<p>
 
<p>Our predicted sequence created in SnapGene<p>
−
<p>insert T--Georgia State--pCB302gfpMBD.png<p>
+
<img src="https://static.igem.org/mediawiki/2019/6/6b/T--Georgia_State--pCB302gfpMBD.png"></img>
 +
</div>
 +
<div class="col-6">
 
<p>The actual plasmid map<p>
 
<p>The actual plasmid map<p>
−
<p>insert T--Georgia State--pcbplasmid.png<p>
+
<img src="https://static.igem.org/mediawiki/2019/a/ab/T--Georgia_State--pcbplasmid.png"></img>
 +
</div>
 +
</div>
 +
 
 +
<p><b><u>pCB302-GFP-MBD Binary Vector</b></u></p>
 +
 
 +
<p>pCB302 is a plant targeted plasmid containing gfp encoding fusions with a microtubule-binding domain (MBD) . pCB302-GFP-MBD Binary Vector is harbored in Agrobacterium tumefaciens to evaluate the heterologous expression of the photosynthetic dinoflagellate, Symbiodinium mircoadriaticum. The plasmid was derived from Dr. Boris Voigt from The Institute for Cellular and Molecular Botany, University of Bonn, Germany who donated it to the Instituto de Biotecnología, UNAM (Cuernavaca, Morelos, México) who then donated it to iGEM GSU.
 +
After pCB302-GFP-MBD in Agrobacterium tumefaciens was introduced into the Symbiodinium mircoadriaticum, the cells lost their photosynthetic pigments and were left unable to reproduce. A successful transformation of the Symbiodinium mircoadriaticum, done by UNAM,  was measured by GFP expression frequency measured as green fluorescence emission yielding 640 per every 1,000,000 cells. Despite losing photosynthetic ability, pCB302-GFP-MBD Binary Vector is the first step to a stable Symbiodinium mircoadriaticum transformation (Ortiz-Matamoros, 2015).</p>
  
−
<p>pCB302-GFP-MBD is a Binary Vector<p>
+
<dl>
 +
<dt>LB- LB T-DNA repeat</dt>
 +
<dd>Left border repeat from nopaline C58 T-DNA</dd>
  
−
<p>pCB302 is a plant targeted plasmid containing gfp encoding fusions with a microtubule-binding domain. pCB302-GFP-MBD Binary Vector is harbored in Agrobacterium tumefaciens to evaluate the heterologous expression of the photosynthetic dinoflagellate, Symbiodinium mircoadriaticum, and created by Instituto de Biotecnología, UNAM (Cuernavaca, Morelos, México).<p>
+
</dl>
−
<p>After pCB302-GFP-MBD in Agrobacterium tumefaciens was introduced into the Symbiodinium mircoadriaticum, the cells lost their photosynthetic pigments and were left unable to reproduce. The successful transformation of the Symbiodinium mircoadriaticum was measured by GFP expression frequency measured as green fluorescence emission yielding 640 per every 1,000,000 cells. Despite losing photosynthetic ability, pCB302-GFP-MBD Binary Vector is the first step to a stable Symbiodinium mircoadriaticum transformation (Ortiz-Matamoros, 2015).<p>
+
  
 +
<dl>
 +
<dt>OriV- incP</dt>
 +
<dd>Origin of replication</dd>
  
−
<p>LB- LB T-DNA repeat<p>
+
</dl>
−
<p>Left border repeat from nopaline C58 T-DNA<p>
+
−
<p>TGGCAGGATATATTGTGGTGTAAAC<p>
+
  
 +
<dl>
 +
<dt>nptIII-</dt>
 +
<dd>Kanamycin resistance gene</dd>
  
−
<p>OriV- incP origin of replication<p>
 
−
<p>AGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCACCCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAAAACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGCGGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCTGTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACTCTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGGCCGCGGTGTCTCGCACACGGCTTCGACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGAGGGCAACCAGCCCGGTGAGCGTC<p>
 
  
−
<p>nptIII- Kanamycin resistance gene<p>
+
<dl>
−
<p>CTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCAT<p>
+
<dt>TrfA-</dt>
 +
<dd>Gene for trans-acting replication protein that binds to and activates OriV</dd>
  
 +
<dl>
 +
<dt>MBD- Microtubule binding domain</dt>
 +
<dd>of the mammalian microtubule-associated protein 4 (MAP4) gene</dd>
 +
</dl>
  
−
<p>TrfA- the gene for trans-acting replication protein that binds to and activates OriV
+
<dl>
−
CTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGGCGTGTTCCACCAGGCCGCTGCCTCGCAACTCTTCGCAGGCTTCGCCGACCTGCTCGCGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTCCAGCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATAGTCGAACATCCGTCGGGCCGTCGGCGACAGCTTGCGGTACTTCTCCCATATGAATTTCGTGTAGTGGTCGCCAGCAAACAGCACGACGATTTCCTCGTCGATCAGGACCTGGCAACGGGACGTTTTCTTGCCACGGTCCAGGACGCGGAAGCGGTGCAGCAGCGACACCGATTCCAGGTGCCCAACGCGGTCGGACGTGAAGCCCATCGCCGTCGCCTGTAGGCGCGACAGGCATTCCTCGGCCTTCGTGTAATACCGGCCATTGATCGACCAGCCCAGGTCCTGGCAAAGCTCGTAGAACGTGAAGGTGATCGGCTCGCCGATAGGGGTGCGCTTCGCGTACTCCAACACCTGCTGCCACACCAGTTCGTCATCGTCGGCCCGCAGCTCGACGCCGGTGTAGGTGATCTTCACGTCCTTGTTGACGTGGAAAATGACCTTGTTTTGCAGCGCCTCGCGCGGGATTTTCTTGTTGCGCGTGGTGAACAGGGCAGAGCGGGCCGTGTCGTTTGGCATCGCTCGCATCGTGTCCGGCCACGGCGCAATATCGAACAAGGAAAGCTGCATTTCCTTGATCTGCTGCTTCGTGTGTTTCAGCAACGCGGCCTGCTTGGCCTCGCTGACCTGTTTTGCCAGGTCCTCGCCGGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGTGTCGATGGTCATCGACTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAACGCTCCACGGCGGCCGATGGCGCGGGCAGGGCAGGGGGAGCCAGTTGCACGCTGTCGCGCTCGATCTTGGCCGTAGCTTGCTGGACCATCGAGCCGACGGACTGGAAGGTTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTCGGCATCCTCGGCGGAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCTTCCGGTCAAACGTCCGATTCAT<p>
+
<dt>GFP- Green fluorescent protein;Plant optimized</dt>
 +
<dd>Reporter gene</dd>
 +
<dd>Binds to microtubules in vitro when fused to MBD</dd>
 +
</dl>
  
 +
<dl>
 +
<dt>Primers for PCR</dt>
 +
<dd>5’-GATGAGTAAAGGAGAAGAAC-3’</dd>
 +
<dd>5’-TAT TTGTATAGTTCATCCATGCC-3’</dd>
 +
</dl>
  
 +
<dl>
 +
<dt>Tnos-</dt>
 +
<dd>Nopaline Synthase Terminator and poly(A) signal</dd>
  
−
<p>MBD- Microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene<p>
+
<dl>
 +
<dt>Bar-</dt>
 +
<dd>Phosphinothricin acetyl transferase</dd>
 +
<dd>Confers resistance to bialophos or phosphinothricin </dd>
  
−
<p>“The MBD contains a proline-rich region (P) and a region that is abundant in serine and proline (SP) that together bind strongly to microtubules and induce their bundling. The remainder of the domain is occupied by degenerate repeats (PGGG) that enhance microtubule binding, probably by stabilizing assembled tubulin heterodimers”(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC143949/pdf/101927.pdf)<p>
+
<dl>
 +
<dt>Pnos-</dt>
 +
<dd>Nopaline Synthase Promoter</dd>
 +
<dl>
 +
<dt>RB- RB T-DNA repeat</dt>
 +
<dd>Right border repeat from nopaline C58 T-DNA</dd>
  
−
<p>GFP- Green fluorescent protein;Plant optimized<p>
 
−
<p>Reporter gene<p>
 
−
<p>Binds to microtubules in vitro when fused to MBD<p>
 
−
<p>ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGTAA<p>
 
  
−
<p>Tnos- Nopaline Synthase Terminator and poly(A) signal<p>
+
<dl>
−
<p>GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC<p>
+
<dt>Forward Primer:</dt>
  
−
<p>Bar- (BlpR)<p>
 
−
<p>Phosphinothricin acetyl transferase<p>
 
−
<p>Confers resistance to bialophos or phosphinothricin<p>
 
−
<p>ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATCTGA<p>
 
  
−
<p>Pnos- Nopaline Synthase Promoter<p>
+
<dl>
−
<p>GAACCGCAACGATTGAAGGAGCCACTCAGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGCGTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCCAAATAATCTGCA<p>
+
<dt>Reverse primers:</dt>  
 +
<dd>5’-GCACTAGTGTAACGACCAATACCCCA-3’</dd>
 +
</dl>
  
−
<p>RB- RB T-DNA repeat<p>
 
−
<p>Right border repeat from nopaline C58 T-DNA<p>
 
−
<p>GTTTACCCGCCAATATATCCTGTCA<p>
 
  
 
<p>Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693<p>
 
<p>Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693<p>
 +
</div>
 +
</div>
 +
</div>
 +
</section>
 +
</div>
 
</div>
 
</div>
 
<!-- Goodbye Area Start -->
 
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Latest revision as of 03:39, 22 October 2019

Composite Part

Composite Parts

Composite Parts
Dino III GFP Plasmid
  • Part:BBa_K3042005
    		•
    Dino III RFP Plasmid
  • Part:BBa_K3042006
    		•
    pCB302-gfp-MBD plasmid
    *This part is not in the registry*

    pCB302-gfp-MBD Plasmid

    Our predicted sequence created in SnapGene

    The actual plasmid map

    pCB302-GFP-MBD Binary Vector

    pCB302 is a plant targeted plasmid containing gfp encoding fusions with a microtubule-binding domain (MBD) . pCB302-GFP-MBD Binary Vector is harbored in Agrobacterium tumefaciens to evaluate the heterologous expression of the photosynthetic dinoflagellate, Symbiodinium mircoadriaticum. The plasmid was derived from Dr. Boris Voigt from The Institute for Cellular and Molecular Botany, University of Bonn, Germany who donated it to the Instituto de Biotecnología, UNAM (Cuernavaca, Morelos, México) who then donated it to iGEM GSU. After pCB302-GFP-MBD in Agrobacterium tumefaciens was introduced into the Symbiodinium mircoadriaticum, the cells lost their photosynthetic pigments and were left unable to reproduce. A successful transformation of the Symbiodinium mircoadriaticum, done by UNAM, was measured by GFP expression frequency measured as green fluorescence emission yielding 640 per every 1,000,000 cells. Despite losing photosynthetic ability, pCB302-GFP-MBD Binary Vector is the first step to a stable Symbiodinium mircoadriaticum transformation (Ortiz-Matamoros, 2015).

    LB- LB T-DNA repeat
    Left border repeat from nopaline C58 T-DNA
    OriV- incP
    Origin of replication
    nptIII-
    Kanamycin resistance gene
    TrfA-
    Gene for trans-acting replication protein that binds to and activates OriV
    MBD- Microtubule binding domain
    of the mammalian microtubule-associated protein 4 (MAP4) gene
    GFP- Green fluorescent protein;Plant optimized
    Reporter gene
    Binds to microtubules in vitro when fused to MBD
    Primers for PCR
    5’-GATGAGTAAAGGAGAAGAAC-3’
    5’-TAT TTGTATAGTTCATCCATGCC-3’
    Tnos-
    Nopaline Synthase Terminator and poly(A) signal
    Bar-
    Phosphinothricin acetyl transferase
    Confers resistance to bialophos or phosphinothricin
    Pnos-
    Nopaline Synthase Promoter
    RB- RB T-DNA repeat
    Right border repeat from nopaline C58 T-DNA
    Forward Primer:
    Reverse primers:
    5’-GCACTAGTGTAACGACCAATACCCCA-3’

    Ortiz-Matamoros, M. F., Islas-Flores, T., Voigt, B., Menzel, D., Baluška, F., & Villanueva, M. A. (2015). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. PloS one, 10(7), e0132693. doi:10.1371/journal.pone.0132693

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