Principles of methods of characterization
Congo Red
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.
Crystal Violet Assay
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]
Fig 1. Crystal violet and Congo Red reagent.
TEM
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.
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.
Fig 2. TEM device at the National Center for Protein Science Shanghai.
Quantum Dots Binding Assay
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).
PLAN 1
Construction of CsgA-Histag
CsgA-HisTag is a part from the previous year IGEM competition. It is documented by team TU_Delft with the Part ID
BBa_K1583003. 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.
Characterization
1. Congo Red：Successful biofilm secretion and expression
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.
Fig 3.Congo red assay of CsgA-Histag on YESCA plates
The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg ml-1 of aTc produced amyloid structures which are dyed red by CR in comparison to the negative control after 72h culture at 30℃. This assay indicates the success in expression of the self-assembly curli fibers.
2. Crystal Violet Assay： Quantification test of biofilm
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.
Fig 4.Crystal violet assay of CsgA-Histag.
3.Quantum dots fluorescence test: successful binding test of Histag with nanomaterials (CdSeS/CdSe/ZnS core/shell quantum dots)
New characterization of the PART BBa_K1583003
After confirming that our parts success in biofilm expression, we are going to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles. We apply suspended QDs solution into M63 medium which has cultured biofilm 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 QDs are attached with biofilms, thus show bright fluorescence. Therefore, we ensure 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.
Fig 5. Fluorescence test of CsgA-His binding with nanomaterials
4. TEM: visualization of binding test
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
Fig 6.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.
Finally, transmission electron microscopy(TEM) visualize the microscopic binding effect of CsgA-Histag fused biofilm with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. From the first picture, it shows biofilm areas are densely covered by CdS nanorods. As can be clearly seen from the second figure, with inducer, there’s distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. Thus we ultimately confirm the viability of bio-abiotic hybrid system.
Fig 7.Representative TEM images of biotemplated CdS nanorods on CsgA-His.
Construction of His-CsgA-SpyCatcher-Histag/ His-CsgA-SpyCatcher
PARTS：BBa_K2132001
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
Linkage System. 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:
BBa_K2132001
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).
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[4].
Characterization
Since the sequence is actually a fusion protein, we identify each unit individually in characterization.
1. Congo Red：Successful biofilm secretion and expression
His-CsgA-SpyCatcher-Histag
After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc and cultured for 72h at 30℃ 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.
Fig 8. Congo Red Assay of His-CsgA-SpyCatcher-Histag
His-CsgA-SpyCatcher
After 72h culture, we scratched 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.
2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials
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.
Fig 9. Congo Red Assay of His-CsgA-SpyCatcher
His-CsgA-SpyCatcher-Histag
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) through 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.
Fig 10. Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.
His-CsgA-SpyCatcher
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.
Fig 11. Quantum dots templating assay on His-CsgA-SpyCatcher biofilm.
3. TEM: visualization of binding test
TEM further characterize the biofilm expressed by strains secreted His-CsgA-SpyCatcher-Histag (HSCH) and His-CsgA-SpyCatcher (HSC) respectively. The distinct nanofiber network manifests the large biofilm expression.
Fig 12. aTc induced secretion of His-CsgA-SpyCatcher-Histag and His-CsgA-SpyCatcher visualized by TEM. Without the presence of inducer, there’s no nanofiber formation around scattered bacteria.
CsgA-His can interface with different inorganic materials since they form the coordinate bonds with the same ligand, Co-NTA, on nanomaterials. Here we use to AuNPs in place of quantum dots and nanomaterials to characterize the validity of Histags on CsgA fused amyloid protein and meanwhile prove the versatility of our biofilm-based platform. As the figures shown, we confirm the feasibility of our newly constructed biobricks to template inorganic material and thus form bio-abiotic hybrid system.
Fig 13. After aTc induced, biofilm secreted by His-CsgA-SpyCatcher-Histag and His-CsgA-SpyCatcher mutants organize AuNP around the cells. In contrast with the third one without inducer, there’s nothing templating on the seemingly smooth outermembrane of bacteria.
