Difference between revisions of "Team:ShanghaitechChina/IBS"

 
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<a href="#Potential use for wider application">Potential use for wider application</a>
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        <h1 align="center">Connection to Project</h1>
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        <h1 align="center" style="font-weight:bold">Abstract</h1>
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<p><strong>Artificial photosynthesis represents a promising solution for energy issues, however, the efficiency, robustness, and scalability does not meet the requirements of industrial applications.</strong> We proposed and demonstrated a sun-powered biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that could potentially solve the issues above. Biofilm-anchored nanorods can efficiently convert photons to electrons, which seamlessly tap into the electron chain of engineered strain carrying FeFe hydrogenase gene cluster, thereby achieving high-efficiency hydrogen production. Furthermore, the intrinsic adherence of biofilms towards various interfaces allows us to grow biofilms on easy-separation micro-beads, therefore facilitating recyclable usage of the biofilm-anchored NRs and endowing this whole system with recyclability. Notably, our hydrogen production has shown great stability compared to some precursors using hydrogenase. Practically speaking, the system comprising <em>E. Coli</em> and biofilms are both amenable for scalable operation, rendering itself a great potential for large-scale industrial applications. Such system can also be adapted to other energy-oriented applications by utilizing engineered new strains with a diverse spectrum of enzymes or metabolic pathways.<strong> The efficiency, recyclability, stability, scalability, and versatility makes our system a design that is truly applicable.</strong></p>
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Biofilms function as a platform to sustain the whole system in vitro. Biofilm-anchored nanorods can efficiently convert photons to electrons, which transfer to engineered strain producing FeFe hydrogenase gene cluster, thereby achieving high-efficiency in biohydrogen production. In addition, a brilliant traits,the intrinsic adherence of biofilms towards various interfaces, allows us to grow biofilms on easy-separation micro-beads. Based on those merits, biofilm stand out by facilitating recyclable usage of the biofilm-anchored NRs and endowing this whole system with recyclability. <p></p>
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<center><img src="https://static.igem.org/mediawiki/2016/5/5d/Plan_1_V2.jpg" style="width:52%"></center>
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        <h2 align="center" style="font-weight:bold">Hydrogenases Expression and Enzyme Activity Assay</h2>
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        <h3>(1) Principles and Methods</h3>
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        In the activity assay of the hydrogenase in producing hydrogen, we repeated three  parallel experiments to test the activity and validated the repeatability of our rudimentary system. In each parallel experiment, the system goes through three periods of “light-on and light-off”. The results (see below) shows the stability of the system and the reversible catalytic activity of the hydrogenase of the reaction, 2H<sup>+</sup> + 2e<sup>-</sup>  ⇿ H<sub>2</sub> .<p></p>
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The three parallel systems consist of <em>E. coli</em> with engineered hydrogenase (wet weight 100ug) resuspended in PBS, 200ul quantum dots/nanorods (7.72*10^-9 M) resuspended in PBS, 150Mm NaCl, 100mM VitaminC, and mediator solution (5mM Paraquat dichloride, for mediating the electrons across the cell membrane). The whole solution including bacteria is adjusted to pH=4 by 100mM Tris-HCl(pH=7.0), given that the pH of 4 was reported to be an optimal environment.<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature. The whole system is based on former study. <span style=”font-size:12px”></span><p></p>
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In addition, we did a fourth assay with resuspended microspheres covered with quantum dots/nanorods bound biofilm in PBS, in place of the resuspended quantum dots/nanorods solution. The fourth set is the actual system we are proposing, since it is as efficient and allows the recycling of quantum dots/nanorods.<p></p>
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In our experiment, we find that despite the reported affected catalytic ability of FeFe hydrogenase due to oxygen, non-strict anaerobic and short-term exposure to oxygen does not cause detrimental effects on the enzyme activity of producing hydrogen. This can be explained by the high catalytic ability and the segregation layer from the atmosphere provided by the hydrogen it produces. Meanwhile, the electron sacrificial agent VitaminC also adds to the “protection layer” of the hydrogenase in our system.<p></p>
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<h3>(2) Instrument</h3>
 
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          <h1 align="center">Introduction</h1>
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<center><img src="https://static.igem.org/mediawiki/2016/0/06/T--ShanghaitechChina--Hydrogenase--chanqingzhuangzhixijie.png" style="width:72%"></center>
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              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.<p></p>
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However, we view biofilms through different lenses to transform those ill impacts into merits.  We envision to establish the Solar Hunter system on E.Coli’s biofilm. Biofilms 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. <p></p>
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What’s more, biofilm can automatically grow by static adherence, which facilitates 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.[1]
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        <h1 align="center">Motivation</h1>
 
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              For the reasons above, biofilms become 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 inorganic nanomaterials and enzymes. <p></p>
 
  
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Based on these ideas, we constructed several E.coli strains which secreted:
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<center><p style="text-align:center"><b>Figure 1</b> Apparatus of the hydrogen production assay.</p></center>
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<li> CsgA-Histag </li>
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<li> His-CsgA-SpyCatcher-Histag </li>
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It contains (1)a light source in our hydrogen production assay acting as a substitute for the real sun. (We chose a high-power white LED light, set 28cm away from the reaction container for a even distribution of photons); (2)  an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a date hub; (5) a computer connected to the hub to record the
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data and generate the curve of concentration variation within a period of time. <p></p>
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<p id="AResults" style="margin-bottom:80px"></p>
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<h3 >(3) Results</h3>
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<h4><b>a) Contribution of each component of the hydrogen production system</b></h4>
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The first hydrogen production data using our system is the pink curve (curve 1) in Figure 1. It shows that lighting can induce hydrogen production in a closed system with nano rods (NR), mediator Methyl Viologen, and IPTG-induced bacteria transformed with fused plasmid. To prove that every element of the system is necessary and that it is our hydrogenase that produced the hydrogen rather than NR, we conducted a series of experiments.<p></p>
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To see whether NR is necessary and whether the hydrogen is produced by the reaction between NR and water under lighting rather than our hydrogenase, we conducted the experiment where the system does not contain nano rods or contain only nano rods. The data is summarized in Figure 1A. The red curve (curve 2) represents the system with the transformed bacterial suspension but without nano rods (NR). The flat curve shows that the system without NR could not produce hydrogen with light; NR is necessary for the system. The black curve (curve 3) represents a system in which only NR and mediators are present, with no bacteria. The flat curve shows that it could not produce hydrogen, which proves that the elements of the bacteria is necessary in the synthesis of hydrogen.<p></p>
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<center><img class="pic3x full" src="https://static.igem.org/mediawiki/2016/b/b1/T--ShanghaitechChina--asasy-conditon--success.png"></center>
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<p style="text-align:center"><b>Figure 2</b></p>
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<center><h3>click to enlarge the figure</h3></center>
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Hydrogen production evolution curve (Sensor Data/ Hydrogen amount vs Time) with different components. The pink curve (curve 1) in all pictures is the hydrogen production with all the components, nano rods (NR), IPTG induction, and the bacteria transformed with our hydrogenase plasmid. The rest are data with one or two components missing. In particular, data in the integrated picture are categorized into Figure 2A and 2B. Figure 2A shows the system with or without nano rods or with nano rods alone, and Figure 2B represents the system with or without induction. The curve 3 in each of the specific figure is the blank control with not transformed <em>E. coli</em> BL21. This series of experiments show that only when both nano rods (NR) and IPTG-induced transformed bacteria are present can the system produce hydrogen in a stable way.<p></p>
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Another step in proving that it is that the hydrogenase is indeed responsible for hydrogen production is to contrast the production level between the induced and un-induced bacteria suspension. The experiment we conducted are summarized in Figure 6B In this set of experiment, the blue line (curve 4) acts as our blank control. In this group, we use the wild type BL21 cells without plasmid. Although we can see a positive oscillation during a short time in the curve, the production was not at high rate and is likely due to the native hydrogenase in <em>E. coli</em>. The green curve (curve 5) represents the transformed bacterial with no induction of IPTG after 12h cultivation. The flat curve shows that it could not produce hydrogen, which proves that the induction of the hydrogenase expression is necessary. To further confirm, we did another experiment using bacteria that have grown 36 hours with no induction. The purple curve (curve 6) clearly contrasts the induced BL21 and the non-induced one. With curve 4 to 6, we have demonstrated that, with the help of NR, it was our hydrogenase in the system that produced the hydrogen we detected.<p></p>
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<h4><b>b) Bidirectional catalytic property of [FeFe] hydrogenase</b></h4>
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As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen (<sub>2</sub>). Thus, when we “turn off” the production mode, we should be able to see the consumption of hydrogen by hydrogenase. In testing this bidirectional catalytic property, conducted an experiment where we turned on and turned off the light alternately. The data is shown below in Figure 3. During lighting period, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of the consumption of hydrogen. It is noteworthy that the hydrogenase shows the greatest production rate at the beginning of lighting: a transient sharp rise can be observed at the valleys. It is also obvious that each period of “light-on light-off” gives similar curves, which implies that our hydrogenase is stable.
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<center><img src="https://static.igem.org/mediawiki/2016/a/ab/T--ShanghaitechChina--asasy--bidirectlycat.png"></center>
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        <p style="text-align:center"><b>Figure 3</b> Verifying the bidirectional catalytic property of [FeFe] hydrogenase.</p>
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        During the period under lighting, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of the consumption of hydrogen.<p></p>
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<h4><b>c) Hydrogen production with nano rods suspension replaced by nano rods bound to biofilm beads.</b></h4>
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Given the difficulty in recycling the nano rods due to their small size, we utilize biofilm to immobilize nano rods and aggregate them into larger assemblies that allow filtration or other ways of recycling including centrifugation. However, testing whether the NR aggregate work in our system is needed. We conducted experiments with nano rods suspension replaced by nano rods bound to biofilm beads. The biofilm, whose subunit was CsgA engineered with HisTag on N-termial and SpyCachter-HisTag on C-terminal, was grown on microspheres, 25 micrometers in diameter for 48 hours. NR’s were then added and given 30 min to bind to the HisTag on CsgA subunit. (The engineered SpyCatcher was used for future pure hydrogenase binding.) The solution was centrifuged and the sediments contained biofilm beads covered with NR. This sediment was resuspended in PBS and was added to the reaction system. The data is in Figure 3 In this experiment, we did the same “light-on light off” actions to the system and the pattern is similar to the one with NR suspension (Figure 2) During lighting, the rapid production of hydrogen can be clearly observed. Some other characteristics pertain, such as the sharp rise at the beginning of lighting.<p></p>
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        <p style="text-align:center"><b>Figure 4</b> Hydrogen production with nano rods suspension replaced by nano rods bound to biofilm beads.</p>
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        We replaced the nanorods suspension with nano rods bound to biofilm beads. During the period with lighting, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of the consumption of hydrogen, as in Figure 3.
 
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We planned two ways to utilize the biofilm display to establish the whole biohydrogen platform:<p></p>
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<b>Comparing Figure 2 and Figure 3</b><p></p>
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<h4><b>Plan 1. Strains secrected CsgA-Histag or His-CsgA-SpyCatcher-(Histag) biofilms for binding nanorods + Strain producing hydrogenase HydA</b></h4>
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In the process of hydrogen generation, a stir bar with a necessary speed of 800 RPM was used to generate the curve in Figure 3. But in Figure 4, a stir bar was not used. It is likely because the aggregates of NR have a bigger chance in colliding with <em>E. coli</em> to transfer electrons. We therefore propose this model as our final model, although further optimization of the system is still under way.
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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. <p></p>
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<h4><b>d) Repeatability</b></h4>
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<h4><b>Plan 2. Strain secreted His-CsgA-Spycatcher-(Histag) biofilms for binding nanorods + purified  hydrogenase HydA-SpyTag</b></h4>
 
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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.<p></p>
 
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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.<p></p>
 
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        <h1 align="center">Key Achievements</h1>
 
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        Above all, we tested and proved that all the strains we constructed work well:<p></p>
 
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1.Strains with engineered CsgA subunits :
 
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can successfully expressed, secreted and realized self-assembly extracellularly.<p></p>
 
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2.Small peptide histag on CsgA subunits can function well and attach to the ligands on nanorods and quantum dots.<p></p>
 
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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.
 
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        <h1 align="center">Mechanism</h1>
 
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              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 facilitates 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. <p></p>
 
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We constructed a library of CsgA biobricks (see <a href="https://2016.igem.org/Team:ShanghaitechChina/Parts">Parts</a>) 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 (<a href="#p6">Results and Optimization</a>) 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.<p></p>
 
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        <h1 align="center">Construction and Characterization</h1>
 
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        <h3>Principles of methods of characterization</h3>
 
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        <h4><b>Congo Red</b></h4>
 
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Congo Red dye is a classic method to detect amyloid protein [2]. Amyloid can be visualized and quantified through the staining of Congo Red because Congo Red molecules obtain an oriented arrangement on amyloid fibrils. This property can be ascribed to the hydroxyl groups on the amyloid and hydrogen bonding on the Congo Red [3]. It only takes approximately 20 minutes to dye so it is indeed a good practice in lab to crudely test the expression of biofilms.<p></p>
 
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Crystal violet is a triarylmethane dye used as a histological stain to classify biomass. This is a simple assay practical and useful for obtaining quantitative data about the relative quantity of cells which adhere to multi-wells cluster dishes. After solubilization, the amount of dye taken up by the monolayer can be quantitated in a plate reader. [4]<p></p>
 
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<p style="text-align:center"><b>Fig. </b> Crystal violet and Congo Red reagent.</p>
 
  
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        <h4><b>TEM</b></h4>
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<center><img src="https://static.igem.org/mediawiki/2016/7/76/T--ShanghaitechCHina--repeat.png"></center>
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In order to visualize the formation and different appearance of biofilm nanowire network, we utilize transmission electron microscope to directly look into the microscopic world. TEM can visualize nano-structure with the maximal resolution of 0.2nm which is beyond the range of optical microscope.  
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In using TEM, samples must be prepared accordingly. The first step is to apply UAc on objects. After object is covered by UAc, the certain area would absorb or cause scattering of electrons and therefore the detector cannot receive transmissive electrons through copper grid, thus leaving a dark shadowy appearance of sample in the image.<p></p>
 
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<p style="text-align:center"><b>Fig. </b> TEM device at the National Center for Protein Science Shanghai.</p>
 
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        <h4><b>Quantum Dots Binding Assay</b></h4>
 
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Mechanisms of Quantum dots binding assay have been introduced in detail in Quantum Dots part. we utilizing Co/Ni-NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) to bind with the histidine in Histags on our biofilm and thus characterize its formation. The whole linkage is performed by forming firm coordinate bonds. They could be applied to quick detection of biofilm expression of His-tagged proteins with naked eye under UV light owing to the photoluminescence of QDs, and accurate concentration measurement under fluorescence spectrum (A detailed protocol for repeatable measurements is included in our Wikipage). <p></p>
 
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        <p style="text-align:center"><b>Figure5.</b>  Repeatability of our hydrogen production system. The three curves conform. This demonstrates our preliminary prototype as repeatable and robust. </p>
  
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To further prove our system as a reliable one, we did three sets of hydrogen production assays in one day in a row (Figure5, curve 10-12). The system was mainly made up of resuspended CdS nonorods, <em>E. Coli</em> BL21 transformed with the plasmid containing all the four [FeFe]hydrogenase subunits from Clostridium. acetobutylicum, and mediator, methyl viologen. (See Figure1 or the section of Principles and Methods for details of the reaction system.) From the data shown, we clearly see the conformation of the three curves. This demonstrates our preliminary prototype in Figure1 as repeatable and robust.
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<h3>Construction of CsgA-Histag</h3>
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CsgA-HisTag is a part from the previous year IGEM competition. It is documented by team TU_Delft with the Part ID <a href="http://parts.igem.org/Part:BBa_K1583003">BBa_K1583003</a>. However, its status not released. Luckily, we obtained the sequence from Allen Chen at Harvard. The two shared the same amino acid sequence, with some difference in the DNA sequence, possibly modified due to the PARTS Standards. We used the Histags on the CsgA-Histag mutant as the binding site of CdS nanorods, meanwhile, we applied methods described previously to characterize CsgA.<p></p>
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<h3 id="Characterization">Characterization</h3>
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<h4><b>1. Congo Red：successful biofilm secretion and expression</b></h4>
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The series of Congo Red assay are aim to visualize the expression of biofilm. To produce curli, we spread the CsgA-Histag mutant E.coli onto a low-nutrition culture medium, YESCA- CR plates[1], containing 10 g/l of casmino acids, 1 g/l of yeast extract and 20 g/l of agar, supplemented with 34 μg /ml of chloromycetin,  5 μg/ ml of Congo Red and 5 μg/ ml of Brilliant Blue. (Details in protocol 链接) Red staining indicates amyloid production.<p></p>
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<p style="text-align:center"><b>Fig.</b>Congo red assay of CsgA-Histag on YESCA plates</p>
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The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg/ ml of aTc will produce amyloid structures which are dyed to red by CR in comparison to the negative control. This assay indicates the success in expression of the self-assembly to curli fibers. <p></p>
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<h4><b>2. Crystal Violet Assay： quantification test of biofilm </b></h4>
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Further, we use crystal violet assay to simply obtain quantitative information about the relative density of cells and biofilms adhering to multi-wells cluster dishes. As illustrated in pictures, CsgA-Histag mutant distinguishes itself in absorbance after applying standard crystal violet staining procedures (See protocal ) in comparison to strain ΔCsgA and 30% acetic acid negative control. There’s certain amount of background absorption of strain ΔCsgA because the dye can stain the remaining E.coli adhering to the well. This difference between induced strains secreted CsgA-Histag and ΔCsgA manifest a distinct extracellular biofilm production in the modified strain. <p></p>
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<p style="text-align:center"><b>Fig.</b>:Crystal violet assay of CsgA-Histag.</p>
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<h4><b>3. Quantum dots fluorescence test:  binding test of Histag with nanomaterials</b></h4>
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<b>new characterization of the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1583003">PART BBa_K1583003</a></b><p></p>
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In order to test the effect of binding between CsgA-Histag mutant and inorganic nanomaterials, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilms for 72h. After 1h incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and  secreted biofilm, and firmly attached with QDS and thus show bright fluorescence. Therefore, we ensured the stable coordinate bonds between CsgA-Histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow. The picture was snapped by ChemiDoc MP,BioRad, false colored.<p></p>
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<p style="text-align:center"><b>Fig.</b> Fluorescence test of CsgA-His binding with nanomaterials</p>
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<h4><b>4. TEM: visualization of binding test</b></h4>
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Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. The first image illustrates biofilm areas which are densely covered by QDs after induced for 72h and incubated, compared to the second image which is not incubated with nanoparticles CdSe. The third one is a negative control without inducer, bacteria scattered without forming biofilm<p></p>
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<img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:100%;">
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<b>Fig.</b>:Representative TEM images of biotemplated  CdSe quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are densely attached to biofilm.
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Finally, transmission electron microscopy(TEM) visualize the binding effect of CsgA-Histag mutant E.coli with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. As can be clearly seen from the figures, with inducer there’s distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. From the first picture, it shows biofilm areas organize CdS nanorods around the bacteria and we confirm the viability of bio-abiotic hybrid system.<p></p>
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<img src="https://static.igem.org/mediawiki/parts/e/e1/Shanghaitechchina_CsgAHistag%2Bnanorods.png" style="width:100%;">
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<p style="text-align:center"><b>Fig.</b>Representative TEM images of biotemplated  CdS nanorods on CsgA-His. </p>
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<h3>Construction of His-CsgA-SpyCatcher-Histag/ His-CsgA-SpyCatcher</h3>
 
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<b>PARTS：<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a></b><p></p>
 
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In light of the immunization platform of biofilm for enzymes, we need some tags acting like glues or stickers that could be connected to the tags on the enzyme. The SpyCatcher and SpyTag system seem like a good choice for us. The SpyCatcher on the biofilm will mildly bind the SpyTag on the enzyme. Note that there is no the other way around, given that the huge size (138 amino acids) may impair the normal function of some delicate enzyme, hydrogenase in our case. For more details for the principles of SpyCatcher and SpyTag and our motivation on this system, see <a href="#p5">Linkage System</a>.  On top of the linkage to the enzyme, we would like to equip the biofilm the ability to bind nanorods and quantum dots. This goal makes the construction of His-CsgA-SpyCatcher-Histag or His-CsgA-SpyCatcher necessary. The two sequences are submitted as our first two original parts. See webpage of the parts here: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a><p></p>
 
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In constructing the sequence, we simply used Gibson Assembly to assemble the clips of CsgA, SpyCatcher, Histag and the plasmid backbone together at one single reaction. For more details and the experiment data, please download the pdf here(此处设置超链接).<p></p>
 
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In constructing the parts, we had been worried about whether the huge SpyCatcher will interfere with the CsgA secretion and whether they will secret together. Careful characterization of each subunit proves that the two parts work excellently, in consistence with previous findings. (Citation) <p></p>
 
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<h3>Characterization</h3>
 
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Since the sequence is actually a fusion protein, we identify each unit individually in characterization.<p></p>
 
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<h4><b>1. Congo Red：successful biofilm secretion and expression</b></h4>
 
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<b>His-CsgA-SpyCatcher-Histag</b><p></p>
 
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After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg/ ml of aTc successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR, compared to the negative control with no inducer.  (Because the ratio between Congo Red dye and Brilliant Blue dye is not in the best state which leads to the unapparent phenomenon through the lens, the brown red biofilm is easy to be identified visually.) This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.<p></p>
 
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<img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width:60%;align:center">
 
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<p style="text-align:center"><b>Fig.</b> Congo Red Assay of His-CsgA-SpyCatcher-Histag</p>
 
  
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<b>His-CsgA-SpyCatcher</b><p></p>
 
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After 72h culture, we scratch the biofilm down from the well and apply 25 μg/ ml of Congo Red into solution. Then centrifuged and washed by PBS for several times, we get the result: newly His-CsgA-SpyCatcher mutant induced by 0.25 μg ml-1 of aTc was stained to bright-red color by CR, compared to the negative control with no inducer and the color can’t be washed away. This assay also manifested the success in construction of His-CsgA-SpyCatcher mutant and add versatility to our biofilm platform.<p></p>
 
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<h4><b>2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials</b></h4>
 
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Then comes to the next part: we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify is the fluorescence assay of binding with nanomaterials. <p></p>
 
 
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<img src="https://static.igem.org/mediawiki/parts/c/c8/Shanghaitechchina_hisCsgASpyCatcherHistag_CR.png" style="width:100%;">
 
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<p style="text-align:center"><b>Fig.</b> Congo Red Assay of His-CsgA-SpyCatcher</p>
 
 
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<b>His-CsgA-SpyCatcher-Histag</b><p></p>
 
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After applying the same steps as introduced above, the bottom of left well show a large area of bright fluorescence, manifesting His-CsgA-SpyCatcher-Histag mutant secreted biofilms under the control of inducer and Histags on it is not blocked by SpyCatcher protein. What is more, it is firmly attached with inorganic materials (i.e.quantum dots) thtough ligand. From this assay, we assure that the SpyCatcher will not impose negative effect on the binding between nanomaterial and biofilm. The picture was snapped by ChemiDoc MP, BioRad, false colored.<p></p>
 
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<img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" style="width:60%;">
 
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<p style="text-align:center"><b>Fig.</b> Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.</p>
 
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<b>His-CsgA-SpyCatcher</b><p></p>
 
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Using the same approach, we also conducted binding assay of His-CsgA-SpyCatcher with QDs to characterize the expression of biofilm and  the visual result shows vividly that His-CsgA-SpyCatcher can bind successfully with the QDs with the existence of inducer aTc, which shows the functional similarity in CsgA-Histag. The picture was snapped by BioRad ChemiDoc MP, false colored.<p></p>
 
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<img src="https://static.igem.org/mediawiki/parts/4/45/Shanghaitechchina_hisCsgASpyCatcher%2BQD.png" style="width:60%;">
 
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<p style="text-align:center"><b>Fig.</b> Quantum dots templating assay on His-CsgA-SpyCatcher biofilm.</p>
 
  
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         <h1 align="center">Linkage System</h1>
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         <h1 align="center" >Potential use for wider applications</h1>
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<b> Our biofilm system included a SpyCatcher system which has the potential for working directly with enzymes on biofilm. This design was not utilized in our hydrogen production so far, but it offers a gate for enzymes to directly react with nanomaterials since the CsgA subunit is engineered with both the Histag and SpyCatcher. This potential use might lead to a boost in efficiency in some nanomaterial-enzymes combinations. The reasoning of the design, and the proof of the functional design is shown below.  </b><p></p>
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               <h3 >SpyTag and SpyCatcher [5]</h3>
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               <h3 class="bg" >SpyTag and SpyCatcher [3]</h3>
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<h4><b>Introduction and Motivation</b></h4>
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<h4><b>Introduction and Motivation: SpySystem</b></h4>
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We want to attach enzyme to biofilm, so we turn to 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 [6]. 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. <p></p>
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We want to attach enzymes to biofilm, so we turn to a widely applied linkage system, SpyTag and SpyCatcher.<p></p>
 
<center>
 
<center>
 
<img src="https://static.igem.org/mediawiki/parts/c/c5/Shanghaitechchina_spy1.png" style="width:50%;">
 
<img src="https://static.igem.org/mediawiki/parts/c/c5/Shanghaitechchina_spy1.png" style="width:50%;">
 
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<h4><b>Motivation and Design</b></h4>
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<h4><b> Design</b></h4>
 
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 subunits and enzyme, and successfully prove their feasibility and stability.<p></p>
 
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 subunits and enzyme, and successfully prove their feasibility and stability.<p></p>
 
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<h4><b>Characterization</b></h4>
 
<h4><b>Characterization</b></h4>
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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. <p></p>
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As Figure6 illustrated, His-CsgA-SpyCatcher-Histag 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 <i>E. coli</i>. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system. <p></p>
 
<center>
 
<center>
 
<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;">
 
<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;">
 
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<p style="text-align:center"><b>Fig. </b>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.</p>
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<p style="text-align:center"><b>Fig 6. </b> 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.</p>
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The new CsgA mutants we obtained or newly constructed, and applied in our Solar Hunter project are as follows:
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<li> CsgA-Histag </li>
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<li> His-CsgA-SpyCatcher-Histag </li>
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<li> His-CsgA-SpyCatcher </li>
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<p></p>           
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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 ) 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. <p></p>
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Nanoparticle binding assay of all constructs library. CdSeS/ZnS core/shell QDs which emit red fluorescence under 365nm UV light 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 1h 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 .<p></p>
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<b>Fig. </b>Nanomaterial binding test. Images were shot by iPhone 5s under 365nm UV light, Tanon UV-100
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We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg/ ml 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 of aTc just reach the optimal point.<p></p>
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[1] Neel S. Joshi, P. Q. (2014, September 17). Programmable biofilm-based materials from engineered curli nanofibres. nature communications.<p></p>
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<p>[1] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H<sub>2</sub> Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.</p>
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[2] 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.<p></p>
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<p>[2] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H<sub>2</sub>, Production of a Whole-Cell Reaction by Recombinant <em>Escherichia coli</em>, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016</p>
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[3] Puchtler, H. S. (1962). On the binding of Congo red by amyloid. Cytochem. <p></p>
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<p>[3] Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).</p>
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[4] Soares, M. J. (n.d.). Crystal Violet Assay. Retrieved from KU MEDICAL CENTER: http://www2.kumc.edu/soalab/LabLinks/protocols/cvassay.htm<p></p>
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[5] Zsofia Botyanszki, 1. P. (2015, May 20). Engineered Catalytic Biofilms: Site-Specific Enzyme Immobilization onto E. coli Curli Nanofibers. Biotechnology and Bioengineering.<p></p>
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[6] Bijan Zakeria, J. O.-L. (2012, February 24). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. PNAS.<p></p>
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Latest revision as of 02:10, 20 October 2016

igem2016:ShanghaiTech