Team:Macquarie Australia/Protocols/ArrivalofGBlocks

From 2012.igem.org

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<html><h1><center>gBlocks Synthesized</h1></center>
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==='''Ordered gBlock fragments are nearly here!'''===
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<p>With our gBlocks fully synthesized by IDT DNA, the BioBricks could be produced. radiodurans bacteriophytochrome. Our gBlocks were codon optimised for <i>E. coli</i> and the physical data and sequences can be seen below,</p>
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<blockquote><ul><li><a href="/wiki/images/c/cc/AgroT7A.pdf" class="internal" title="AgroT7A.pdf">+T7 promoter Agrobacterium tumefaciens bacteriophytochrome </a>
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</li><li><a href="/wiki/images/8/8a/AgroA.pdf" class="internal" title="AgroA.pdf">-T7 promoter Agrobacterium tumefaciens bacteriophytochrome </a>
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</li><li><a href="/wiki/images/d/d4/T7Heme.pdf" class="internal" title="T7Heme.pdf">+T7 promoter Heme oxygenase </a>
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</li><li><a href="/wiki/images/a/a0/HemeA.pdf" class="internal" title="HemeA.pdf">-T7 promoter Heme oxygenase </a>
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</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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<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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Soon our gBlock fragments will arrive from IDT. On Tuesday (4/09/2012) we will perform Gibson assembly will assemble the following four biobricks:
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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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* [[media:AgroT7A.pdf | +T7 promoter Agrobacterium tumefaciens bacteriophytochrome ]]
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<p>Using these gBlock BioBricks will be synthesised following this <a href="https://2012.igem.org/Team:Macquarie_Australia/Protocols/GibsonAssembly">procedure</a>.
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* [[media:AgroA.pdf | -T7 promoter Agrobacterium tumefaciens bacteriophytochrome ]]
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</html>
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* [[media:T7Heme.pdf | +T7 promoter Heme oxygenase ]]
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* [[media:HemeA.pdf | -T7 promoter Heme oxygenase ]]
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Click on each above to see the corresponding fragment nucleotide sequence and fragment properties.  
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<br>
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{| class="wikitable"
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|-
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! scope="col"| BioBrick name
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! scope="col"| Fragment(s)
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|-
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! scope="row"| T7 Heme oxygenase
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| Hemo_T7_A + Hemo_B + Plasmid
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|-
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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 Agro bacterium
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| Agro_T7_A + Agro_B + Agro_C + Agro_D + Agro_E + Plasmid
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|-
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! scope="row"| Agrobacterium (T7 minus)
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| Agro_A + Agro_B + Agro_C + Agro_D + Agro_E
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|}
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Latest revision as of 23:57, 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.

Using these gBlock BioBricks will be synthesised following this procedure.

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