Difference between revisions of "Part:BBa K3470023"

Latest revision as of 16:11, 26 October 2020

Usage and Biology

A his-tag, or polyhistidine tag, is a string of histidine residues at either the N or C terminus of a recombinant protein. There can be from four to ten residues in a string, although commonly there are six histidine residues a hexahistidine tag. Some recombinant proteins are engineered to have two hexahistidine tags. His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. The most common ion for his-tag purification of a recombinant protein is Ni 2+ , though Co 2+ , Cu 2+ , and Zn 2+ are also used. The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Most other proteins in the lysate will not bind to the resin, or bind only weakly. The use of a his-tag and IMAC can often provide relatively pure recombinant protein directly from a crude lysate.

Proposed Experimentation

To estimate the activity of the dual enzyme system, MerA and MerB must be purified using his-tag protein purification protocol and concentration must be measured (from each circuit).

Circuits:

1.Constitutive Promoter – RBS – MerA - His-tag - Double Terminator

2.Constitutive Promoter – RBS – MerB - His-tag - Double Terminator

3.Constitutive Promoter – RBS – MerA - His-tag – RBS - MerB-His-tag - Double Terminator

Different concentrations of each enzyme must be mixed into different test tubes containing 2.9µM and 3.8µM of methylmercury (chloride) (for each concentration of enzyme) in 5mL water. At 60 min intervals, 1mL of the mixture must be pipetted out and the concentration of mercury must be mapped using gas chromatography.

Three graphs must be plotted: -Methylmercury concentration vs time keeping enzyme concentration constant. -Enzyme concentration vs activity graph -Number of cells vs enzyme concentration graph.

Using the graphs we can estimate the optimal amount and enzyme required to be produced for optimal activity. We can also estimate the optimum CFU/mL to be used to get maximum activity.

Note: According to the results obtained in the MATLAB model, the optimal concentration of enzymes must be in the order 10-4.

References

[1] Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews, 27(2–3), 355–384. https://doi.org/10.1016/S0168-6445(03)00046-9

[2] Kimple, M. E., Brill, A. L., & Pasker, R. L. (2013). Overview of affinity tags for protein purification. Current protocols in protein science, 73, 9.9.1–9.9.23. https://doi.org/10.1002/0471140864.ps0909s73

[3] Mathema, V. B., Thakuri, B. C., & Sillanpää, M. (2011). Bacterial mer operon mediated detoxification of mercurial compounds: A short review. Archives of Microbiology, 193(12), 837–844. https://doi.org/10.1007/s00203-011-0751-4

[4] Parks, J. M., Guo, H., Momany, C., Liang, L., Miller, S. M., Summers, A. O., & Smith, J. C. (2009). Mechanism of Hg-C protonolysis in the organomercurial lyase MerB. Journal of the American Chemical Society, 131(37), 13278–13285. https://doi.org/10.1021/ja9016123