This year we set out to lay down fundamental design principles for surface display systems. To accomplish this, automation enabled a systematic approach to finding optimal combinations of passengers, displayers and spacer elements. Several characteristic passengers (described in previous sections) varying in size and function were examined to give a clearer picture of the spectrum of functional display systems achievable with our approach. The results of our investigations support the idea that more complex passengers are much more difficult to display than simpler ones. Additionally, the inclusion of spacer elements appears to enhance display of passengers to a large degree. Finally, it is interesting to note that although one displayer may be ideal for the functional display of one passenger, it is not necessarily the best display system for all passengers. This makes it increasingly helpful to have a systematic approach to find the best displayer-spacer match for the passenger of interest.
Following completion of functional assays of the passenger-display systems, each quantitative data set was normalized to an appropriate control. The heat map below compiles this data, enabling further analysis of the display systems. For each passenger system, the constructs with the highest signal of functionality were assigned a value of one (white), whereas those with as much as (or less) activity as the control were assigned a value of zero (black). Those displayer-passenger systems for which no data was collected are labeled with blue.
The heat map above points to an interesting trend made clear by the streptavidin and mgfp-5 data. Although all constructs contain short linkers between the displayers and passengers, the inclusion of spacer elements for both systems appears to enhance functional surface display of the passengers. Moreover, the identity of the spacer element is an important parameter determining display efficiency. There is an increase in functional display when the INP repeats spacer is added between the displayers and the strep tag as is seen in the increase in lighter blocks in the map. This trend is especially evident in the mgfp data in which there are several weak signals (many dark blocks in the map) for mgfp displayed on its own. With the addition of several spacer elements, a significant general increase in signal for almost 100% of the systems is observed.
Design and Future Work
With the data collected for each passenger, quantitative analysis for the design of optimized displayer-passenger systems can be performed. This analysis provides the basis for a probabilistic system for predicting successful display of specific passenger-displayer combinations. In order to enhance the design system developed, we intend to conduct future characterization of a wider variety of passengers, displayers and spacers.
To see our design principles in action, check out our discussion for the design of a new passenger in the 2009 Berkeley iGEM presenation.