Difference between revisions of "Part:BBa K2692000"

Latest revision as of 04:53, 17 October 2018

CutA metal binding protein from E.coli

CutA is a metal binding protein found naturally in Escherichia coli . This protein in the cell ensures that ion homeostasis is retained in the cell, whereby excess of copper ions is removed by this protein [1]. This part contains a Hexahistidine tag for purification purposes.

Figure 1- 2% Agarose gel demonstrating the CutA and Cup1 genes. The samples in the gel are in the following lanes: lane 1- digested PSB1C3; lane 2- PSB1C3; lane 3- CutA plasmid 1; lane 4- CutA plasmid 2; lane 5- CutA plasmid 3; lane 6- Cup1 plasmid 1; lane 7- Cup1 plasmid 2; lane 8- Cup1 plasmid 4; lane 9- Cup1 plasmid 5; lane 10- 1Kb ladder from Bio Basic; lane 11- CutA PCR 1; lane 12- CutA PCR 2; lane 13- CutA PCR 3; lane 14- Cup1 PCR 1; lane 15- Cup1 PCR 2; lane 16- Cup1 PCR 3; lane 17- Cup1 PCR 4.

Figure 1 shows our cloning results of CutA and Cup1 where we ligated the coding sequence into the pSb1C3. At this stage they are not expressable. However, being that we were able to get the CutA gene in a expressible plasmid (pCA24N), we were able to purify the CutA protein with nickel affinity chromatography and size exclusion chromatography.

Figure 2 - 15% SDS-PAGE of Nickel affinity batch purification of CutA. In the first lane we used the molecular weight marker RMR002 from GMbiolab. Lanes 2-5 show the elutions that contain our Cut A protein. CutA protein runs at around 12kDA; however, in our SDS-PAGE gel it is seen at around 14kDa, this is likely because of the histidine tag. The remaining lanes are as follows: 6- Nickel Regeneration; 7- Cell Lysate Before Binding; 8- Cell Lysate After Binding; 9- Wash Sample; 10- Cell Pellet.

Figure 3 - 15% SDS-PAGE of Size Exclusion Chromatography elution samples. The first column contains the molecular weight marker RMR002 from GMbiolab, and lanes 2-16 contain our elutions.

Figure 4- The chromatogram of the size exclusion Chromatography purification. After purification we were able to try an assay were we tried to determine if our protein was able to bind copper effectively.

Figure 5- The standard curve for the binding assay, where the absorbance values of standard copper sulfate concentrations were determined. From there, concentrations of copper bound could be determined.

Figure 6- The results from the copper assay.

The results suggest that there is a specific incubation time and concentration of copper sulfate that was optimal for activity. However, this data was found to be insignificant and that too much copper and too little protein was used during the assay. Details on our results can be found at http://2018.igem.org/Team:Lethbridge_HS/Results.

Sequence and Features