Team:Macquarie Australia/Protocols/ArrivalofGBlocks

From 2012.igem.org

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</li><li><a href="/wiki/images/a/a0/HemeA.pdf" class="internal" title="HemeA.pdf">-T7 promoter Heme oxygenase </a>
</li><li><a href="/wiki/images/a/a0/HemeA.pdf" class="internal" title="HemeA.pdf">-T7 promoter Heme oxygenase </a>
</li><li><a href="/wiki/images/c/cf/DeinoA.pdf" class="internal" title="DeinoA.pdf">Deinococcocus radiodurans bacteriophytochrome</a>
</li><li><a href="/wiki/images/c/cf/DeinoA.pdf" class="internal" title="DeinoA.pdf">Deinococcocus radiodurans bacteriophytochrome</a>
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</li></ul></blockquote>
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<br>
<br>
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<center><table class="wikitable">
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<tr>
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<th scope="col"> BioBrick name
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</th><th scope="col"> Fragment(s)
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</th></tr>
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<tr>
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<th scope="row"> T7 Heme oxygenase
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</th><td> Hemo_T7_A + Hemo_B + Plasmid
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</td></tr>
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<tr>
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<th scope="row"> Heme oxygenase (T7 minus)
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</th><td> Hemo_A + Hemo_B + Plasmid
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</td></tr>
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<tr>
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<th scope="row"> T7 Agrobacterium
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</th><td> Agro_T7_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
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</td></tr>
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<tr>
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<th scope="row"> Agrobacterium (T7 minus)
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</th><td> Agro_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
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</td></tr>
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<tr>
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<th scope="row"> Deinococcus radiodurans bacteriophytochrome
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</th><td>Deino_A + Deino_B + Deino_C + Deino_D + Deino_E + Plasmid</td></tr></table></center>
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{| class="wikitable"
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<p>The Agrobacterium bacteriophytochrome and heme oxygenase genes were constructed with the T7 promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I719005">Registry Part BBa_I719005</a>). Due to the large sequence of the D. radiodurans bacteriophytochrome, the fragments will be assembled without a T7 promoter. We had experienced delays in receiving our gBlock fragments due to high GC content in the designed fragments which would have caused problems during synthesis. A high GC content results in the production of hairpin loops which terminates synthesis and thus we had to reduce the GC content. By reducing the GC content of certain fragments, we were forced to change the 30 bp overlap sequence in order for these sequences to overlap during Gibson Assembly. At one stage, Deino_A contained 80% GC content and thus had to be altered in order for synthesis by IDT to begin.</p>
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! scope="col"| BioBrick name
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! scope="col"| Fragment(s)
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! scope="row"| T7 Heme oxygenase
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| Hemo_T7_A + Hemo_B + Plasmid
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! scope="row"| Heme oxygenase (T7 minus)
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| Hemo_A + Hemo_B + Plasmid
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! scope="row"| T7 Agrobacterium
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| Agro_T7_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
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! scope="row"| Agrobacterium (T7 minus)
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| Agro_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
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Once the Deinococcus Radiodurans fragments arrive we will assemble them using Gibson assembly. Due to the large sequence of the D. radiodurans bacteriophytochrome, the fragments will be assembled without a T7 promoter. The designed sequences of the 5 fragments can be viewed[[media:DeinoA.pdf | here ]]
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{| class="wikitable"
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! scope="col"| BioBrick name
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! scope="col"| Fragment(s)
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! scope="row"| Deinococcus radiodurans bacteriophytochrome
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| Deino_A + Deino_B + Deino_C + Deino_D + Deino_E + Plasmid
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We had experienced delays in receiving our gBlock fragments due to high GC content in the designed fragments which would have caused problems with hairpin loop formation and thus we had to reduce the GC content. By reducing the GC content of certain fragments, we were forced to change the 30 bp overlap sequence in order for these sequences to overlap during Gibson Assembly. At one stage, Deino_A contained 80% GC content and thus had to be altered in order for synthesis by IDT to begin.
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Revision as of 23:53, 22 September 2012



gBlocks Synthesized

With our gBlocks fully synthesized by IDT DNA, the BioBricks could be produced. radiodurans bacteriophytochrome. Our gBlocks were codon optimised for E. coli and the physical data and sequences can be seen below,


BioBrick name Fragment(s)
T7 Heme oxygenase Hemo_T7_A + Hemo_B + Plasmid
Heme oxygenase (T7 minus) Hemo_A + Hemo_B + Plasmid
T7 Agrobacterium Agro_T7_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
Agrobacterium (T7 minus) Agro_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
Deinococcus radiodurans bacteriophytochrome Deino_A + Deino_B + Deino_C + Deino_D + Deino_E + Plasmid

The Agrobacterium bacteriophytochrome and heme oxygenase genes were constructed with the T7 promoter (Registry Part BBa_I719005). Due to the large sequence of the D. radiodurans bacteriophytochrome, the fragments will be assembled without a T7 promoter. We had experienced delays in receiving our gBlock fragments due to high GC content in the designed fragments which would have caused problems during synthesis. A high GC content results in the production of hairpin loops which terminates synthesis and thus we had to reduce the GC content. By reducing the GC content of certain fragments, we were forced to change the 30 bp overlap sequence in order for these sequences to overlap during Gibson Assembly. At one stage, Deino_A contained 80% GC content and thus had to be altered in order for synthesis by IDT to begin.

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