Difference between revisions of "Team:ASU/Results"

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.

Arsenate reductase (ArsC) and ferritin (FtnA) were introduced separately into our plasmid backbone MT-pASapI via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 5-alpha competent e.coli cells from NEB. The cloning was successful, as the positive control plate for ArsC and FtnA showed significantly more colonies than the negative control plates. This was confirmed with a restriction digest of the plasmid using XbaI and BstxI. FtnA was expected to have two bands: 4.4kb and 2.7 kb, while ArsC was expected to have two bands of 4.4kb and 2.6kb. This is a contrast to the MT-pASapI backbone, which would result in two bands of 4.4kb and 2.3kb The gel showed three bands for all of the constructs run, the top band being undigested plasmid due to ineffective restriction digest. However, the other bands showed that the inserts contained bands of the appropriate size, and were different from the MT plasmid.

The tagged sequences ArsC-Ha, MT-6xHIS, and FtnA-FLAG were introduced separately into our plasmid backbone MT-pASapI via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent e.coli cells from NEB. The cloning was successful, as there were more colonies on the positive control plates than on the negative controls. However, this was confirmed via restriction digest of the plasmid using BstXI and AflII. MT-6xHIS was expected to have 3 bands: 4.8kb, 1.3kb, and 809bp. ArsC-HA was expected to have two bands: 5.8kb and 1.3kb. FtnA-Flag was expected to have 2 bands: 5.9 kb and 1.3kb. The number and size of bands revealed from the gel were as expected, indicating that the plasmid had been successfully integrated with our tagged inserts, with a few exceptions that were noted.

The combinations MT-FtnA, MT-ArsC, and ArsC-FtnA were ultimately cloned into the MT-pASapI backbone via golden gate assembly. The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent e.coli cells from NEB. The cloning was ultimately successful, after a few iterations and design changes. For more information about the iterative design process, see our Engineering Success page! The inserted sequences were confirmed via restriction digest using XbaI and BstxI. MT-FtnA digest was expected to have two bands of 4.4kb and 2.4kb. MT-FtnA was expected to have two bands of 4.4kb and 3.2kb. MT-ArsC was expected to have two bands of 4.4kb and 3.7kb. The number and size of bands shown in the gel were as expected, and indicated that the plasmid had been successfully integrated with our combined parts.

The “Full” sequence, MT-IEE2-ArsC-IEE5-FtnA, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. For more information, see our Engineering Success page. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent e.coli cells.