igem2016:ShanghaiTech

Biofilm Introduction

Biofilms are ubiquitous as 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. We envision to establish the Solar Hunter system on E.Coli’s biofilm. Biofilm can substantially increase the resistance of bacteria to adverse conditions like acid or oxidative stress and form a stable and balanced system. These traits can elevate its adaptability to application to industry for they do not need to be meticulously taken care of and are capable to withstand harsh conditions. Therefore, it will be a good practice to reduce the production cost.

What’s more, biofilm can automatically grow by static adherence, which facilitate s 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. Also, it has been tested that CsgA subunits fused with not too large peptide can be tolerated by curli export machinery and maintain the self-assembly function as always. (Neel S. Joshi, 2014)

Motivation

For the reasons above, biofilm becomes our best candidate to engineer and would be equipped with some additional functions we want. Here, we conceive the semiconductor-enzyme system linked to the 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.

Based on these ideas, we constructed:

CsgA-Histag
His-CsgA-SpyCatcher-Histag
His-CsgA-SpyCatcher

We envision two ways to utilize the biofilm display to establish the whole biohydrogen platform:

Plan 1. Strains secrected CsgA-his or His-CsgA-SpyCatcher-(Histag) biofilms for binding nanorods + Strain producing hydrogenase HydA

Through this approach, we want to realize producing hydrogen by attach nanorods onto biofilms. The electrons from nanorods excited by sunlight can transfer into engineered hydrogenase-producing strain through mediator solution and accepted by hydrogenases which are not secreted. Since anaerobic hydrogenase will not be exposed to oxygen directly in this way, we view it as a practical and promising way to conduct in lab and consequently realize biohydrogen.

Plan 2. Strain secreted His-CsgA-Spycatcher-(Histag) biofilms for binding nanorods + purified hydrogenase HydA-SpyTag

Based on this concept, we want to construct a catalytic system outside cells. After extracted and purified from strain which produce hydrogenase, the HydA-Spytag engineered enzyme could covalently bind with SpyCatcher protein on the Strain secrected His-CsgA-Spycatcher-(Histag) biofilms. At the meanwhile, nanorods are firmly attach to biofilm as well for there are histags on biofilm subunits. Electrons from nanorods excited by light thus transfer directly to purified HydA due to short spacial distance and achieve hydrogen production in vitro.

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.

Mechanism

We focused on the bacterial amyloid curli structure. The curli consists of two kinds 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 s extension of nanowire. CsgA is about 13-kDa, whose transcription needs 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(Neel S. Joshi, 2014). CsgA subunits secreted by different bacteria individuals will not have trouble in assembling and bridging each other, therefore finally achieving the goal as extensive as an organized community network.

We constructed a family of CsgA biobricks (see Parts) which are respectively modified with different small peptide domain, endowing the biofilm with designed functions. The expression of CsgA is strictly controlled by inducer anhydrotetracycline (aTc) and its biomass can be tuned by the concentration of inducer (see later for pictures) so that the biofilm is only formed when we need it and is conductive to be well operated when our system is industrialized. Next, we demonstrate the experiments we conducted to test the expression, quantify the biomass, and analyze the viability of different CsgA biobricks.

Construction and Characterization

Linkage System

SpyTag and SpyCatcher (Zsofia Botyanszki, 2015)

A widely applied linkage system, SpyTag and SpyCatcher, originally discovered from Streptococcus pyogenes. By splitting its fibronectin-binding protein FbaB domain, we obtain a relatively small peptide SpyTag with 13 amino acids and a bigger protein partner, SpyCatcher, with 138 amino acids (Bijan Zakeria, 2012). 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 condition (room temperature), thus set lower requirement for reaction both in lab and future practice. 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.

Characterization

As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicating the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system.

**Fig.** :The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria.. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.

Team:ShanghaitechChina/Biofilm