"

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
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.