For this year’s iGEM competition, our team has chosen two new parts to finish our project.
The first one is Nanobody with aldehyde-based precursor label, can be converted to fixed-point aldehyde-modified Nano (BBa_K2772000). It is anti-beta2 microglobulin nanobody with aldehyde-based precursor label, and it can be converted to fixed-point aldehyde-modified Nanobody by formylglycine-generating enzyme (FGE). It contains two sections: nanobody and ald tag, which is precursor of aldehyde label. Firstly it can cambein with beta2 microglobulin because of antigen-antibody interaction. Then its special label allow fixed point fixing or other fixed point decoration possible.
Another one is Formylglycine-generating enzyme(BBa_K2772018). FGE can specifically identify ald-Tag, which is consisted of 6 base pair or 13 base pair, then it oxidze sulfhydryl group of sulfhydryl group to aldehyde group. Conveniently, it can be used for in vivo catalysis of the substrate label of the precursor, i.e. higher conversion can be achieved without purification separation.
Here is the list of our submitted parts.
|BBa_K2772000||Nanobody with aldehyde-based precursor label||528bp|
We used SPR (Surface plasmon resonance) to characterize Nanobodies.
First, we immobilized the antigen bound by our Nanobody on the SPR crystal. Then we configured different concentrations of Nanobody solutions (including blank solutions), and then, through a microflow system, a solution with nanobody is injected over the bait layer. As nanobody binds the antigen bound, an increase in SPR signal (expressed in response units, RU) is observed. After desired association time, a solution without nanobody (usually the buffer) is injected on the microfluidics that dissociates the bound complex between antigen bound and nanobody. Now as nanobody dissociates from the antigen bound, a decrease in SPR signal (expressed in resonance units, RU) is observed. From these association ('on rate', ka) and dissociation rates ('off rate', kd), the equilibrium dissociation constant ('binding constant', KD) can be calculated. The equilibrium dissociation constant represents the strength of our affinity.
After calculation, our KD is 9.127×10-8.