Team:ShanghaitechChina/Biofilm

• • Biofilm introduction
  • Motivation
  • Mechanism
  • Construction and Characterization
  • Linkage System
  • Results and Optimization
  • Achievements
  • For Fun

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.

Fig. 1:Big Picture of Biofilm Part.

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.

Results and Optimization

The new CsgA mutants we obtained or newly constructed, and applied in our Solar Hunter project are as follows:

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

Crystal Violet Assay of the peptide fusion mutants library transformed into ΔCsgA strains. Firstly, we apply 0.1% crystal violet solution to all mutants to gain quantitative data about their relative expression and secretion performance. The result was read and exported by BioTek CYTATION5. CsgA-His mutant stands out as the highest peak in absorbance after washing. (See protocal 链接) The reason why His-CsgA-SpyCatcher-Histag and His-CsgA-SpyCatcher mutant doesn’t perform as well as CsgA-His might be ascribed to size hindrance of SpyCatcher, which impedes the transport process of CsgA mutant subunits from inner area to extracellular environment by CsgG outermenbrane exporter, whose pore size is around 2 nm. Yet, the differences between induced CsgA-His, His-CsgA-SpyCatcher-Histag, His-CsgA-SpyCatcher mutant and negative control exhibit a success extracellular biofilm production in all our constructed and modified strains.

Nanoparticle binding assay of all constructs library. QDs are templated by CsgA-His mutant incubated in M63 solution. We added equivalent amount of QDs solution into M63 medium with mutant E.coli which have been cultured for approximately 72h and all mutants were induced by aTc. After 15min incubation, we apply PBS washing 3 times to wash away the unbinding quantum dots. Pictures demonstrate CsgA-His were produced by three mutants we constructed and QDs are templated on biofilms .

Fig. :Nanomaterial binding test. Images were shot by iPhone 5s under 365nm UV light, Tanon UV-100

We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg ml-1 of aTc will induce the best expression performance of biofilm. The possible reason for higher concentration of inducer strangely leading into low production of biofilm might lie in that aTc, a kind of antibiotic, can be harmful to protein synthesis in bacteria. We speculate there’s an antagonism between the effect of promoting expression and impeding growth brought by aTc and 0.25 μg ml-1 of aTc just reach the optimal point.

Achievements

Above all, we tested and proved that all the strains we constructed work well: 1.Strains with engineered CsgA subunits : 1) CsgA-Histag 2) His-CsgA-SpyCatcher-Histag 3) His-CsgA-SpyCatcher can successfully expressed, secreted and realized self-assembly outside cell membrane. 2.Small peptide histag on CsgA subunits can function well and attach to the ligands on nanorods and quantum dots. 3.Large protein SpyCatcher on CsgA subunits are also able to be secreted by transporter machinery and successfully form nanofibers. We also prove the biological function of SpyCatcher after appending on CsgA subunits, thus provide potential for our second plan mentioned above.

For Fun

We bought a personalized sticky paper with ShanghaiTech University logo. Initially, we paste the sticky paper on the bottom of the plate and add 25ml M63 minimum medium to culture strain secreted CsgA-His. After 72h culture, QDs with red emission light is applied to the plate and incubated for another 24h. Then solution is removed and we visualize the effect under UV light.

Fig. :QDs templating by CsgA-His fusion protein A)Under natural light against white desk B)Under 365nm UV light against black paper in dark room C) In dark room against black paper

Reference

Alan MarcusEvita Sadimin, Maurice Richardson, Lauri Goodell,and Billie Fyfe,. (2012). Fluorescence Microscopy Is Superior to Polarized Microscopy for Detecting Amyloid Deposits in Congo Red–Stained Trephine Bone Marrow Biopsy Specimens. Am J Clin Pathol.

Bijan Zakeria, J. O.-L. (2012, February 24). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. PNAS.

Neel S. Joshi, P. Q. (2014, September 17). Programmable biofilm-based materials from engineered curli nanofibres. nature communications.

Soares, M. J. (n.d.). Crystal Violet Assay. Retrieved from KU MEDICAL CENTER: http://www2.kumc.edu/soalab/LabLinks/protocols/cvassay.htm

Zsofia Botyanszki, 1. P. (2015, May 20). Engineered Catalytic Biofilms: Site-Specific Enzyme Immobilization onto E. coli Curli Nanofibers. Biotechnology and Bioengineering.

Puchtler, H. S. (1962). On the binding of Congo red by amyloid. . Cytochem.