We produced a Heme Oxygenase BioBrick that was codon optimize for E. coli. The Gibson assembly of the T7 promoter containing Heme Oxygenase was successful. The transformation was successful with numerous colonies grown using Chloramphenicol as the selecting agent. Six colonies were selected and then they were sequenced before digestion with EcoR1 and Spe1. The sequencing suggested that all of the colonies contained the plasmid with a Heme oxygenase identical to the original protein sequence. The gel containing the digested Heme Oxygenase bearing plasmid can be seen in Figure 1.

Figure 1: The restriction digest showing the linearised plasmid backbone (Black Box) and the heme oxygenese gene (Green Box). We used a 1kb ladder.

The plasmid was sequencing using the forward and reverse primers for the BioBricks. We performed Blastx pipeline to determine if there was a significant change in the protein sequence.

Given that this was the source of our gene, we proposed that the sequencing result was accurate. We then compared to the original gBlock sequence and determined that the sequencing was accurate and confirmed the identity of the plasmid.

The T7 bearing Heme Oxygenase produced was characterised to determine if it was functional. BL21 E. coli was transformed with the plasmid, selected for using chloramphenicol and a culture was inoculated. The culture was then induced with ALA (d-aminolevulinic acid) for the heme pathway and IPTG to promote protein production. They were incubated overnight and the cells were spun down. We observed a functional Heme Oxygenase and the cells appeared a vibrant green after induction by ALA and IPTG. We observed this as well in our assembled switch. The image below demonstrates the green produced compared to uninduced Heme Oxygenase and the Bacteriophytochrome.

Like the Heme Oxygenase, the bacteriophytochromes from Deinococcus radiodurans and Agrobacterium tumefaciens were optimised for use in E. Coli. The identity of the plasmid was determined by sequencing and by digestion.