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Team:Macquarie Australia/Protocols/ArrivalofGBlocks
From 2012.igem.org
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</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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<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> | <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> | ||
| + | <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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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.
