Biofilm Introduction

We envision to establish the Solar Hunter system on E.Coli’s biofilm. Biofilms are ubiquitous since they can be found both in human and some extreme environments. They can be formed on inert surfaces of devices and equipment, which will be hard to clean and cause dysfunction of the device.

However, we view it through different lenses to transform this ill impact into merits. Biofilm can substantially increase the resistance of bacteria to adverse conditions like acid or oxidative stress and form a stable and balanced system. This traits can elevate its adaptability to application to industry for they don’t need to be meticulously taken care of and capable to withstand harsh conditions. Therefore, it’ll be a good practice to reduce the production cost. What’s more, biofilm can automatically grow by static adherence , which facilitate regeneration and recycling in mass production in industry. Startlingly, biofilms can also serve as a synthetic nonconductive biological platform for self-assembling function materials. The amyloid protein CsgA , which is the dominant component in E.Coli, can be programmed to append small peptide domain and successfully secreted with biological functions. For that reason, biofilm became our best candidate to engineer and would be equipped with the function we want. Here, we conceive the semiconductor-enzyme system based on E.Coli’s biofilm, whose subunits are engineered respectively with PolyHistidine tags and SpyTag and SpyCatcher system from FbaB protein to provide binding sites for quantum dots and enzymes. Our ultimate goal is to harness this bio-abiotic hybrid system to efficiently convert solar energy into alternative energy or other high value-added industrial products.

Motivation

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Mechanism

In order to have a further study of biofilm, we focused on the bacterial amyloid curli structure. The curli consists of two kind of amyloid proteins bound together and extending on the cell membrane. CsgA, the main subunit, can self-assemble in the extracellular space creating an amyloid nanowire while CsgB is the part which anchors to the membrane, nucleating CsgA and facilitate extension of nanowire. CsgA is about 13-kDa, whose transcription need to be regulated by CsgD and expression are processed by CsgE, F and secreted with the assistance of CsgC, G (these all belong to curli genes cluster. After secretion, CsgA assembles automatically to form amyloid nanofibers , whose diameter is around 4-7 nm and length varies (Joshi et al., 2014). CsgA subunits secreted by different bacteria individuals won’t have trouble in assembling and bridging each other, therefore realize to expand as a organized community network.

Linkage System

Spytag and Spycatcher A widely applied linkage system, SpyTag and SpyCatcher, originally discovered from Streptococcus pyogenes. By splitting its fibronectin-binding protein FbaB domain, we gain a smaller peptide SpyTag with 13 amino acids and a bigger protein partner, SpyCatcher, with 138 amino acids. The advantage of this system lies in the following three aspects. Firstly, they can spontaneously form a covalently stable bond with each other which guarantee the viability of the permanent linkage. The second point is quick reaction within 10 min, which will stand out by its efficiency in industrial application. Besides, the whole process proceeds in mild conditions(room temperature), thus set lower requirement for reaction both in lab and future application. Therefore, we design to leverage this advantageous system to achieve the binding of biofilm with specific enzyme.

Appending SpyTag to CsgA subunit is a traditional and hackneyed approach to modify biofilm posttranscriptionally. Here, we challenge to attach larger part, SpyCatcher, to CsgA to enrich the versatility of biofilm platform. For one thing, we intend to pioneer new approach. For another aspect is that we concern SpyCatcher is too large that might jeopardize the biological activity and function of the enzyme. After comprehensive consideration, we decide to append SpyTag and SpyCatcher respectively to CsgA subunit and enzyme, and successfully prove their feasibility and stability.

Construction and Characterization

The series of Congo Red assay are aim to ensure the expression of biofilm. To produce curli, we spread the CsgA-histag mutant E.coli onto a low-nutrition culture medium, YESCA- CR plates, containing 10 gl-1 of casmino acids, 1 gl-1 of yeast extract and 20 gl-1 of agar, supplemented with 34 ug ml-1 of chloromycetin, 25 ug ml-1 of Congo Red and 5 ug ml-1 of Brilliant Blue. ( Details in protocol 链接)