What we do
This year, we are developing a project which utilizes three orthogonal connection systems to detect enzyme-catalyzed reaction products qualitatively and quantitatively.
Although enzymes can be used in purified form, in crude cell lysates, or encased in synthetic protective materials, an approach that optimally combines the criteria of high surface area, enhanced enzyme stability, rapid mass transport, and modularity remains elusive. Recently, a brand-new protein immobilization platform has been explored. The platform modifies curli nanofibers, the amyloid fiber component of E. coli biofilms, with a peptide tag fused to the amyloidogenic protein CsgA.
Meanwhile, a genetically programmable, irreversible immobilization method—the spontaneous covalent bond formation between 13-amino acid SpyTag and 15kDa SpyCatcher has been demonstrated. CsgA with a fused SpyTag and a certain enzyme with a SpyCatcher fusion are proved to form site-specific attachment between tags and catchers, even in a complex mixture.
In our approach, we are going to transform PHL628-△csgA cells with pBbE1a plasmids expressing CsgA-SpyTag to cover cells with curli fibers as the base. Curli fibers will connect adjacent cells to form a biofilm(Fig.1). Then, since there are three orthogonal systems, SpyTag/SpyCatcher2, SnoopTag/SnoopCatcher and SdyTag/SdyCatcher , we are also going to synthesizing a string of three enzymes rather than one single SpyCatcher-Enzyme. The method is specifically illustrated as follows(Fig.2).
Where the value lies
One advantage is that it promises to evolve into a cell-free system with high fidelity. It is reported the biofilm displaying the same level of activity after 12 days with a slight decrease after 28 days, which indicates that the biofilm and its entangled curli fibers are stable enough.Thus, a reasonable assumption is that even if cells die, curli with SpyTag and the string of enzymes still works. In view of this advantage, the assembled system has the potential to be widely practiced in industry.
Another advantage is that the string has a myriad of multi-enzyme systems by assigning specific functions to each enzyme at different sites. For example, a logic network composed of three enzymes, alcohol dehydrogenase (ADH, from Saccharomyces cerevisiae), glucose dehydrogenase (GDH, from Pseudomonas) and glucose oxidase (GOx, from Aspergillus niger) operating in concert as four concatenated logic gates(AND/OR) was designed to process 4 different chemical input signals (NAD, acetaldehyde, glucose and oxygen) (Fig.3). The conceived functions of the three enzymes will be discussed later. There is no exaggeration that microelements, such as domains and subunits, provide a possibility to achieve multiple functions.
How hardware assists
It is acknowledged that culture of cells using various microfluidic devices is becoming more common within experimental cell biology. Based on mature technology of microfluidic cell culture, we attempt to control over experimental conditions precisely via custom designed chip architectures, parallelization, automation and direct coupling to miniaturized downstream analysis platforms. This is how purified products, the strings of three enzymes, are produced.
A method using ink-jet printing for constructing multi-enzyme systems was proposed. Considering the vaporization of liquids on ink-jet printers while printing, which may lead to protein denaturation and enzyme inactivation, we decided to transform a 3D printer with similar principles. The capability of 3D printer in creating a precise two-dimensional distribution of enzymes indicates a possibility of spatially controlling enzymatic reactions.
As for the application of our model, it is diversified and remains infinite possibilities. We have already cited one application called enzyme-based logic systems above. Moreover, biochips are preferred. The original intention is to monitor enzyme-catalyzed reaction in real-time with sensitive currents which is generated by the enzymatic biofuel cells (EBFCs) and reflects the product concentration and other indicators.
Adopting nanostructured materials for biofuel cell construction has been extensively suggested as an effective and promising strategy to achieve high energy production; nanotubes can directly connect the enzyme active centers and transport the produced electrons to the electrode along the tubular network. The bacteria growing on the nanotubes enables enzymes on the curli-connected string to close contact nanotubes, which acts as an anode. In addition, metal nanoparticles are similar in size to some enzymes, thus decreasing the electron transfer distance of the bioelectrodes. So we will interface curli fibers with gold nanoparticles(AuNPs) as well to further improve performance of EBFCs(Fig.4). Laccases with electrodes yields the cathode units of EBFCs.
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