Team:Macquarie Australia/Project/Parts

From 2014.igem.org

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<h4> Parts Submitted by Team Macquarie Australia 2014 </h4><br/>
<h4> Parts Submitted by Team Macquarie Australia 2014 </h4><br/>
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</html><groupparts>iGEM014 Macquarie_Australia</groupparts><html>
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<p><table class="tableizer-table">
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<tr class="tableizer-firstrow">
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<th>Part Name</th>
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<th>Type</th>
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<th>Description</th>
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<th>Designer</th>
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<th>Length</th>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326000">BBa_K1326000</td>
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<td>Composite</td>
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<td>Plac + CTH1 + YCF54</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>1700</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326004">BBa_K1326004</td>
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<td>Composite</td>
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<td>Plac + ChlI1 + ChlD</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>3347</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326008">BBa_K1326008</td>
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<td>Composite</td>
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<td>Plac + ChlI1 + ChlD + GUN4</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>4083</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326001">BBa_K1326001</td>
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<td>Coding</td>
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<td>ChlM</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>867</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326002">BBa_K1326002</td>
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<td>Composite</td>
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<td>Plac + CTH1 + YCF54 + Plasto + ChlM</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>2913</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326003">BBa_K1326003</td>
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<td>Composite</td>
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<td>Plac + POR + DVR1 + ChlP + ChlG</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>4615</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326005">BBa_K1326005</td>
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<td>Composite</td>
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<td>Plac + CTH1 + YCF54 + Plasto</td>
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<td>iGEM14_Macquarie_Australia</td>
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<td>2032</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326006">BBa_K1326006</td>
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<td>Composite</td>
 +
<td>Plac + POR + DVR1</td>
 +
<td>iGEM14_Macquarie_Australia</td>
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<td>2250</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1326007">BBa_K1326007</td>
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<td>Composite</td>
 +
<td>Plac + POR + DVR1 + ChlP</td>
 +
<td>iGEM14_Macquarie_Australia</td>
 +
<td>3557</td>
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</tr>
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</table></p>
</div>
</div>
</html>
</html>

Revision as of 15:17, 17 October 2014

Project


Parts & Characterization

The Macquarie 2014 team designed and constructed the following three operons required for the chlorophyll biosynthesis pathway. These three operons have been sent to the registry.

Functional Operons

Operon 1:BBa_K1326008
In anaerobic bacteria, the bch1 and bchD genes are part of an operon. Macquarie 2014 have constructed an equivalent synthetic operon in E. coli, using separate ChlI1 and ChlD taken from oxygenic photosynthetic eukaryotes. We have shown that our artificial operon works, and that proteins self-assemble to form a functional ChlI1:ChlD complex in E. coli. This part works together with ChlH to insert magnesium into protoporphyrin IX. Although our operon does not contain ChlH as originally planned (due to issues in fully assembling the part), in vitro assays indicated full functionality of self-assembly and catalytic functionality.

Figure 1. Operon 1, made up of the lac promoter, ChlI1, ChlD, and GUN4. The BioBrick plasmid backbone encodes chloramphenicol resistance.

Figure 2. Schematic representation of the conversion of Protophoryrin IX to Mg-Protophoryrin IX via Mg-chelatase (Operon 1). The functionality of this step has been demonstrated in our project.

Operon 2:BBa_K1326002
This gene has been used in an operon with other genes responsible for catalysing the biosynthesis pathway from Mg-protoporphyrin IX to Protochlorophyllide. CTH1, Plastocyanin, and YCF54 are involved in the oxidative cyclase pathway. ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. CTH1 catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with YCF54 and Plastocyanin.

Figure 3. Operon 2, made up of the lac promoter, CTH1, YCF54, Plasto, and ChlM. The BioBrick plasmid backbone encodes chloramphenicol resistance.

Operon 3:BBa_K1326003
This gene has been used in an operon with other genes responsible for the terminal steps of the chlorophyll biosynthesis pathway, in the conversion of divinyl protochlorophyllide to chlorophyll a. DVR1 reduces divinyl protochlorophyllide, POR converts protochlorophyllide to chlorophyllide, ChlG adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule, and ChlP reduces the double bonds on GGPP. The final product is chlorophyll a.

Figure 4. Operon 3, made up of the lac promoter, POR, DVR1, ChlP, and ChlG. The BioBrick plasmid backbone encodes chloramphenicol resistance.

The ChlD Story: the repair of the registry ChlD

Team:iGEM2013_Macquarie_Australia designed all 13 parts required for the biosynthesis pathway and successfully synthesized most of these, including ChlD [BBa_K1080002.]. However, this part was not received by the registry in 2013.

We screened and verified the identity of all their parts, as we required them to assemble composite parts with the aim of forming functional operons. When ChlD was sequenced, we determined that there was a 50 base-pair deletion in the middle of the gene. Team:iGEM2014_Macquarie_Australia has repaired this part, ensuring the full correct sequence is present. We incorporated this part it into operon 1, and have verified that the part is functional, guaranteeing it has been properly repaired.

This part has now been successfully sent to the registry in a functional state.

Parts Annotated and Improved

The following parts were again designed and synthesized by Team:iGEM2013_Macquarie_Australia. Many of these were not received by the registry in 2013. Following our efforts to confirm the DNA sequences, we improved their documentation to further elucidate the interactions between each part of the system. Information regarding each part's function, role in the biosynthesis pathway, protein structure, and enzymatic reactions, all of which can be found in the iGEM parts registry.

Parts from previous teams used

Parts Type Description Designer Length
BBa_K1080002 CodingChlD iGEM13_Macquarie_Australia 2154
BBa_K1080003 Coding GUN4 iGEM13_Macquarie_Australia 728
BBa_K1080006 CodingPlastocyanin iGEM13_Macquarie_Australia 324
BBa_K1080000 Coding ChlI1 iGEM13_Macquarie_Australia 1116
BBa_K1080001 Coding ChlH iGEM13_Macquarie_Australia 4125
BBa_K1080005 Coding CTH1 iGEM13_Macquarie_Australia 1152
BBa_K1080007 CodingPORiGEM13_Macquarie_Australia 1067
BBa_K1080008 Coding ChlP iGEM13_Macquarie_Australia 1299
BBa_K1080009 CodingChlG iGEM13_Macquarie_Australia 1050
BBa_K1080010 Coding YCF54 iGEM13_Macquarie_Australia 471
BBa_K1080011 CodingChlI2 iGEM13_Macquarie_Australia 1212
BBa_K1080012 Coding DVR1 iGEM13_Macquarie_Australia 1106
BBa_K1080013 CompositeGUN4 (+ Ptac Promoter) iGEM13_Macquarie_Australia 797
BBa_K1080014 Composite Plasto (+ Ptac promoter) iGEM13_Macquarie_Australia 393
BBa_K1080015 Composite POR (+ Ptac promoter) iGEM13_Macquarie_Australia 1136
BBa_K1080016 Composite ChlI1 (+Ptac promoter) iGEM13_Macquarie_Australia 1185
BBa_K1080017 Composite ChlH (+ Ptac promoter) iGEM13_Macquarie_Australia 4194
BBa_K1080018 Composite ChlD (+ Ptac promoter) iGEM13_Macquarie_Australia 2223
BBa_K1080019 Composite ChlM (+ Ptac promoter) iGEM13_Macquarie_Australia 942
BBa_K1080020 Composite CTH1 (+ Ptac promoter) iGEM13_Macquarie_Australia 1221
BBa_K1080021 Composite ChlP (+ Ptac promoter)iGEM13_Macquarie_Australia 1368
BBa_K1080022 Composite ChlG (+ Ptac promoter) iGEM13_Macquarie_Australia 1119
BBa_K1080023 Composite YCF54 (+ Ptac promoter) iGEM13_Macquarie_Australia 540
BBa_K1080024 Composite ChlI2 (+ ptac promoter) iGEM13_Macquarie_Australia 1281
BBa_K1080025 Composite DVR1 (+ Ptac promoter) iGEM13_Macquarie_Australia 1175
BBa_K864400 Regulatory Ptac, trp & lac regulated promoter iGEM12_Uppsala 61


Parts improved

Part Name Type Description Designer Length
BBa_K1080002 Singular/Coding ChlD iGEM13_Macquarie_Australia 2154

Parts Submitted by Team Macquarie Australia 2014


Part Name Type Description Designer Length
BBa_K1326000 Composite Plac + CTH1 + YCF54 iGEM14_Macquarie_Australia 1700
BBa_K1326004 Composite Plac + ChlI1 + ChlD iGEM14_Macquarie_Australia 3347
BBa_K1326008 Composite Plac + ChlI1 + ChlD + GUN4 iGEM14_Macquarie_Australia 4083
BBa_K1326001 Coding ChlM iGEM14_Macquarie_Australia 867
BBa_K1326002 Composite Plac + CTH1 + YCF54 + Plasto + ChlM iGEM14_Macquarie_Australia 2913
BBa_K1326003 Composite Plac + POR + DVR1 + ChlP + ChlG iGEM14_Macquarie_Australia 4615
BBa_K1326005 Composite Plac + CTH1 + YCF54 + Plasto iGEM14_Macquarie_Australia 2032
BBa_K1326006 Composite Plac + POR + DVR1 iGEM14_Macquarie_Australia 2250
BBa_K1326007 Composite Plac + POR + DVR1 + ChlP iGEM14_Macquarie_Australia 3557

Retrieved from "http://2014.igem.org/Team:Macquarie_Australia/Project/Parts"