Team:Edinburgh/Parts
Parts
Short summary
Our best parts facilitate immobilisation of proteins to silica surfaces, which are beneficial not only for our SuperGrinder concept, but also for a variety of other purposes, as demonstrated throughout our Wiki. For example, our Contribution page demonstrates how silica-tagged proteins can be purified in a quick and cheap protocol using silica spin columns. Our Results section also demonstrates the potential of these tags for development of high-throughput screening for directed evolution purposes.
Best basic part
Immobilisation of sfGFP thanks to L2NC-linker silica tag: BBa_K3946052
The immobilisation efficiency was tested by directly visualising immobilised sfGFP (Figure 1). We found that sfGFP is indeed immobilised via tags we created, namely Car9 (BBa_K3946001), L2NC (BBa_K3946002), and L2NC-linker (BBa_K3946052). We compared fluorescence on beads to the untagged sfGFP, and could observe a residual glowing green colour on the beads incubated with sfGFP with different tags, enabling us to evaluate relative binding efficiencies (Figure 1). Although preliminary results gave problem in the expression of L2NC-linker, later results indicated that L2NC with the addition of a short flexible linker is the silica-binding tag with the highest affinity to silica.
Figure 1 sfGFP immobilised with various silica-binding tags on four silica beads: Celite545, 100um silica beads, glass beads acid washed and glass beads 6 mm.
Silica bound stability test: BBa_K3946052 and BBa_K3946055
The silica-binding tag that provides the strongest enzyme binding is the L2NC-linker tag. Therefore, this experiment was carried out to immobilise the cellulase Cex with L2NC-linker tag (BBa_K3946052) onto silica beads Celite 545 and test the potential of maintaining Cex enzyme activity after immobilisation in a prolonged incubation at 37°C.
Comparing the stability of enzymes from after immobilisation and after 16 hours of incubating, we observed that the initial velocity of Cex changed by 30.76% and 35.49% for free enzyme Cex and cex-L2NC-linker, while only 13.37% changed for the immobilised enzyme (Figure 2). This indicates that Cex is more stable after immobilisation on the silica beads. Thus Cex-L2NC-linker (BBa_K3946055) is our best composite part.
Figure 2 The Cex assay of free enzyme, free enzyme with L2NC-linker tag and immobilised enzyme.