The nature of CINDY Seq is such that, in principle, it can be used to visualize any significant change in polymer length. As a result, this provides a promising outlook to characterize existing and future biobricks that involve the polymerization or degradation of coacervate-forming polymers (see Figure 3). Examples of such enzymes are: nucleases, proteases and amylases. Furthermore, it has been demonstrated that even the molecule ATP can form coacervates (Jia et al. 2014), so that one can even imagine to demonstrate energy consumption or ATP synthesis.
An obvious example of a BioBrick that can directly be characterised using the coacervation detection method is BBa_K1923001, encoding for a variant of Cas13a closely related to the one we have used in our project. With the coacervation detection method any crRNA and RNA target combination can be tested, for activating Cas13a, and even binary tests for off-target effects can be done by introducing mutations in the target RNA.
Figure 3: Schematic outline of the CINDY Seq method. Proteins that can be characterised by CINDY Seq are involved in the assembly and degradation of polymers.
Another example of a BioBrick that could be characterized using coacervates is T5 Phage Exonuclease (BBa_K676006). This part encodes for a DNase that can denature linear ssDNA and dsDNA, but will not cleave supercoiled plasmids, which makes it ideal when manufacturing plasmids. It has been demonstrated that coacervates can also be made using ssDNA and dsDNA (Jain & Vale 2017). The cleaving activity of this (and other) nuclease can therefore be tested using the coacervation detection method. One more nuclease that can characterised in a similar way is the coding sequence for a nuclease regulated by T7-promoter (BBa_K2144000).