Team:Edinburgh/Contribution

The SuperGrinder

Contribution


  • JUMP assembly:
  • We have tested the novel JUMP assembly method which we believe could greatly help other teams to enable a flexible, modular assembly method compatible with Phytobrick standardised parts. We developed a video tutorial on this method.

    Read about JUMP assembly and watch our video on our Communication page.

  • Parts contributions:
  • We have designed and submitted parts compatible with JUMP assembly for the iGEM registry for other teams to use, including signal peptides for protein secretion and silica binding tags for protein immobilisation and purification.

    Find out more about the parts that we have submitted on our Parts page.

  • Advances in protein immobilisation:
  • We have characterised silica-binding tags for protein immobilisation. Silica is a lower cost immobilisation support than many standard lab options, so could be highly useful for other iGEM teams. We encourage other iGEM teams to try it and would be happy to give guidance!

  • Protein purification method development:
  • We also extended our investigation to begin to develop a method for protein purification using silica spin-columns. We developed this protocol using the silica-tagged GFP that we constructed using JUMP assembly, and QIAprep spin columns from our plasmid miniprep kit. This makes the protocol accessible to most other iGEM teams. It is a potentially cost-saving approach compared to standard protein purification methods such as Ni-NTA columns for His-tag chromatography.

    DNA adsorption to the membrane is highly pH dependent, and minimal adsorption is observed above pH 8 (Figure 1). As the optimal affinity for L2 tag is observed around pH 8, using a buffer with pH=8 helps to minimise adsorption of nucleic acids from the crude cell lysate and maximise recovery of tagged proteins. Tagged sfGFP was immobilised to the spin columns after binding and washing twice in 50 mM Tris-HCl (pH 8). L2NC showed highest saturation of binding (Figure 1C & E).


    Silica spin column protein purification protocol

    Figure 1 Silica spin column protein purification. A. DNA recovery (%) from QIAquick membranes under different pH (Figure from QIAgen handbook). B. Spin column purification protocol, showing binding, wash and elution stages. Figure created in BioRender. C. Top view of silica spin columns in UV visualiser after binding (4000g, 2 min) and washing twice in 50 mM Tris-HCl pH=8 (6000g, 1 min), 0.5s exposure. D. Top view of silica spin columns in UV visualiser after elution with 50 mM Tris-HCl pH=8 supplemented with 2 M MgCl2 (4000g, 2 min), 0.5s exposure E. Top view of silica spin columns in natural light after washing and before elution. F. GFP fluorescence of eluted fractions observed in UV visualiser, after elution with 2 M MgCl2.

    Elution was tested using 50 mM Tris-HCl buffer (pH 8) supplemented with 2M MgCl2. Visual analysis suggests that the technique is promising for purification of L2NC and Car9-tagged proteins on a small scale, however as high concentrations of MgCl2 are incompatible with Coomassie assay, protein concentration of eluted fractions could not be measured to evaluate binding capacity of the spin column (without removal of MgCl2 by dialysis). Previous literature suggests that Car9-tagged proteins can be eluted using less severe elution conditions, such as 0.5M L-lysine which could be more compatible with downstream applications. This would be a good future experiment.

    It is unclear why fluorescence of L2 is lost both on membrane and in the eluted fraction, although natural fluorescence is lower which may reduce visual detection. Without SDS-PAGE analysis it is difficult to conclude whether it was successfully eluted.