Team:Calgary/Validation
Xpress ( BBa_K3945014 )
We have utilized Xpress vector to clone in and expression the lanmodulin protein. We initially faced difficulties expressing lanmodulin using the IDT vector but upon being gifted the Xpress vector by Dr. Schryvers we began cloning our lanmodulin construct into this vector. Utilizing Xpress’s ones-step screening method we were able to quicky identify positive colonies containing the inert of interest after just one round of cloning.
Figure 1. One-step screening method using the Xpress vector. Positive colonies show growth only on ampicillin plate (left) while negative colonies also show growth on the kanamycin plate (right).
By streaking the transformed colonies onto a kanamycin and an ampicillin plate we were able to quickly identify which clones have taken up the lanmodulin insert as they were expected to have their kanamycin gene knocked out. The selected positive colonies were later sequenced verified for the right construct. Thus, we have successfully shown that the Xpress’s unique screening technique could be used for efficient cloning of genetic constructs.
Once a GST-LanM fusion construct was obtained in the Xpress vector it was subject to protein production via the autoinduction media. As opposed to the lanmodulin protein, the GST-LanM was easily expressed and purified in E. coli. This was confirmed that by SDS-PAGE analysis - The dark band in lane 9 (purple box) was observed around the 39 kDa range which corresponds to the expected molecular weight of GST-LanM protein (Fig 1). As such we have successfully demonstrated that the GST fusion tag in the Xpress vector could help with efficient production of lanmodulin, a protein that was previously difficult to express.
Figure 2. SDS analysis of his-tagged purified lanmodulin
Lanmodulin ( BBa_K3945001 )
Once we successfully expressed lanmodulin, we tested its REE binding capacity using a metal recovery assay. In this assay an initial known sample of neodymium was incubated with lanmodulin. The protein fraction was then removed and flitrate was analyzed for its neodymium content using Arsenazo assay.
Figure 3. Concentration of neodymium in the initial sample, the filtrate and after treatment with HCl. The samples were treated with either 5 uM of lanmodulin, 5 uM of GST or water.
It was shown that the concentration of neodymium in the filtrate dropped considerably after incubation with lanmodulin, indicating that the protein was able to grab onto most of the neodymium present in the solution. In fact when a percent neodymium recovery was calculated it was determined that lanmodulin recovered more than 80% of the neodymium from the solution whereas GST (negative control) left more than half the metals in the solution. Thus, we have successfully characterized lanmodulin’s extraordinary ability to bind rare earth elements and extract them from the solution.
Figure 4.Percent neodymium recovery efficiency of 5 uM lanmodulin, 5 uM GST and water from a 146 uM initial sample of neodymium.
