Team:BrownStanfordPrinctn/Improve
Broader Functionalization of the Cellulose Surface:
Improving on BBa_K1321340 with BBa_K3260036
Improving on BBa_K1321340 with BBa_K3260036
Improving on BBa_K1321340 with BBa_K3260036
BBa_K1321340, or the N-terminal linker + dCBD submitted by Imperial in 2014, has been widely used as a way to functionalize a cellulose surface. We used BBa_K1321340 as an integral part of our paper microfluidics subproject, but found that trying to fuse too many functional domains to this part led to unsuccessful synthesis. We were able to fuse one domain at a time (BBa_K3260031), but anything more than one additional functional domain attached to the dCBD was not successfully expressed.
We wanted to expand the potential of this part to host a variety of sizes of functional domains. To do this, we developed a split-GFP leucine zipper add-on to BBa_K1321340 that takes the burden off of one protein to host multiple domains, as those domains can be on the other half of the split protein. This system is illustrated in what we have submitted as our improved part, BBa_K3260036, which is complementary to BBa_K3260034.
The system takes the dCBD and the N terminal linker, BBa_K1321340, and attaches a short leucine zipper ZR and one half of a split GFP domain. The burden carried by this construct is similar to that of BB_K1321348, in which a full sfGFP is fused to a dCBD. The design of this construct allows for another construct containing the complementary leucine zipper ZE and the other half of the split GFP domain, in addition to any assortment of functional domains that the project requires. We attached our hydrophobic domain (BBa_K3260025) to this complementary construct to cause water occlusion.
The incorporation of split GFP also allows for a visual reporter of the correct assembly of your constructs.
Below are our results using BBa_K1321340 in the way is was originally intended, and then our improvement upon it with BBa_K3260036. This characterization is also present on the registry pages for
BBa_K1321340:
In our experiments with BBa_K1321340, we took advantage of the endogenous linker and fused a hydrophobic domain to the N-terminus to create the part BBa_K3260031.
BBa_K3260031 Single Layer Print:
The video above shows one of our tests with printing with protein lysate. The lysate that we loaded into the black ink cartridge contained BBa_K3260031(our dCBD + our hydrophobic, or Radek) domain. Directly below is a labelling of this video with channel widths for each component in our testing template.
The blue regions are plain printer ink, while to the white sections in between the blue and the black outline of the channels are printed protein lysate. We can see on the range of straight, separated channels that the water efficiently wicked up the 250um wide channel and the 200um channel. On the largest “flower” template, the water flowed up the 800um and 900um channels. We also see a successful serendipitous negative control of our device once the water flows past the end of one of the 900um channels and starts penetrating the paper printed with blue ink.
This provides a nice contrast with the channel flow and illustrates that while the resolution of the channels isn’t extremely sharp, the dCBD is definitely bound to the cellulose substrate, and the hydrophobic protein is certainly performing its function. On the smaller scale flower channels, the water efficiently travels down the 100 - 500 um channels, which is indicative of its functionality on lower resolution ranges.
The effectiveness of the hydrophobic domain is only made possible by the binding affinity of BBa_K1321340, the dCBD.
BBa_K3260036:
Above is a test of the functionality of two of our new fusion proteins, BBa_K3260036, and BBa_K3260034. If these two parts assemble correctly on the cellulose surface, there should be fluorescence, which is what we observed here. This means that the leucine zipper components of each protein aligned and held together, allowing the two halves of GFP to get close enough together to complete their chromophore and fluoresce. Because we see fluorescence (highlighted in the color swatch of each sample), the success of the leucine zippers and therefore the functionality of the last two functional domains can be inferred as well, the double cellulose binding domain anchoring the whole complex to the cellulose surface, as well as the hydrophobic domain sticking up from the N-terminal region of BBa_K3260034, and therefore the assembled protein complex. The successful assembly of these two biobricks not only shows the applicability of these constructs for paper-based microfluidics, but it also shows the potential of split-GFP reporting and increased functional domain presence for fusion proteins in functional applications.