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Team:Macquarie Australia/Protocols/ArrivalofGBlocks
From 2012.igem.org
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,
- +T7 promoter Agrobacterium tumefaciens bacteriophytochrome
- -T7 promoter Agrobacterium tumefaciens bacteriophytochrome
- +T7 promoter Heme oxygenase
- -T7 promoter Heme oxygenase
- Deinococcocus radiodurans bacteriophytochrome
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
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 here
BioBrick name Fragment(s) Deinococcus radiodurans bacteriophytochrome Deino_A + Deino_B + Deino_C + Deino_D + Deino_E + Plasmid 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.
