Metallothionein (MT) was introduced into our plasmid backbone pASapI via Gibson assembly. This sequence was designed specifically with two type IIS restriction sites for SapI on either side of the MT sequence and ordered from IDT. This allowed us to utilize the MT plasmid as the backbone for all consecutive cloning and enabled us to proceed with golden gate assembly in the future. MT was cloned into 5-alpha competent e.coli cells from NEB. The transformation was successful, as the positive control plate for MT had significantly more colonies than the negative control plate. This cloning was confirmed via restriction digest using XbaI and BstxI. During the Gibson assembly, we excised the NcoI-HindIII linker in the pASapI backbone which contained an XbaI site. Therefore, the restriction digests of MT should only yield two bands (4.4kb, 2.4 kb) instead of the three bands from pASapI (4.4kb, 1.4kb, 0.8kb). The presence of only two bands, the increased size of the second band, indicated that MT was successfully cloned into the plasmid. For all future experiments, this plasmid was used as the backbone, known as MT-pASapI.