Difference between revisions of "Part:BBa K2692000"
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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 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. | ||
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Figure 2 - 15% SDS-PAGE of Nickel affinity batch purification of CutA. | 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. | 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. | ||
Revision as of 02:15, 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 histogram of the size exclusion Chromatography purification.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]