Difference between revisions of "Team:ShanghaitechChina/Biofilm"
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| − | <h3>Principles of methods of characterization</h3> | + | <h3 class="bg">Principles of methods of characterization</h3> |
| − | <h4><b>Congo Red</b></h4> | + | <h4 class="bg"><b>Congo Red</b></h4> |
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> | 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> | ||
| − | <h4><b>Crystal Violet Assay</b></h4> | + | <h4 class="bg"><b>Crystal Violet Assay</b></h4> |
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> | 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 1. </b> Crystal violet and Congo Red reagent.</p> | <p style="text-align:center"><b>Fig 1. </b> Crystal violet and Congo Red reagent.</p> | ||
| − | <h4><b>TEM</b></h4> | + | <h4 class="bg"><b>TEM</b></h4> |
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 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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<p style="text-align:center"><b>Fig 2. </b> TEM device at the National Center for Protein Science Shanghai.</p> | <p style="text-align:center"><b>Fig 2. </b> TEM device at the National Center for Protein Science Shanghai.</p> | ||
| − | <h4><b>Quantum Dots Binding Assay</b></h4> | + | <h4 class="bg"><b>Quantum Dots Binding Assay</b></h4> |
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> | 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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| − | <h3>Construction of CsgA-Histag</h3> | + | <h3 class="bg">Construction of CsgA-Histag</h3> |
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> | 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> | ||
| − | <h3 id="Characterization">Characterization</h3> | + | <h3 id="Characterization" class="bg">Characterization</h3> |
| − | <h4><b>1. Congo Red：Successful biofilm secretion and expression</b></h4> | + | <h4 ><b>1. Congo Red：Successful biofilm secretion and expression</b></h4> |
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> | 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 3.</b>Congo red assay of CsgA-Histag on YESCA plates</p> | <p style="text-align:center"><b>Fig 3.</b>Congo red assay of CsgA-Histag on YESCA plates</p> | ||
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> | 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> | ||
| − | <h4><b>2. Crystal Violet Assay： Quantification test of biofilm </b></h4> | + | <h4 ><b>2. Crystal Violet Assay： Quantification test of biofilm </b></h4> |
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> | 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 4.</b>Crystal violet assay of CsgA-Histag.</p> | <p style="text-align:center"><b>Fig 4.</b>Crystal violet assay of CsgA-Histag.</p> | ||
| − | <h4><b>3. Quantum dots fluorescence test: binding test of Histag with nanomaterials</b></h4> | + | <h4 ><b>3. Quantum dots fluorescence test: binding test of Histag with nanomaterials</b></h4> |
<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> | <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> | ||
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> | 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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| − | <h3>Construction of His-CsgA-SpyCatcher-Histag/ His-CsgA-SpyCatcher</h3> | + | <h3 class="bg">Construction of His-CsgA-SpyCatcher-Histag/ His-CsgA-SpyCatcher</h3> |
<b>PARTS：<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a></b><p></p> | <b>PARTS：<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a></b><p></p> | ||
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> | 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> | ||
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> | 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> | ||
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]. <p></p> | 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]. <p></p> | ||
| − | <h3>Characterization</h3> | + | <h3 class="bg">Characterization</h3> |
Since the sequence is actually a fusion protein, we identify each unit individually in characterization.<p></p> | Since the sequence is actually a fusion protein, we identify each unit individually in characterization.<p></p> | ||
<h4><b>1. Congo Red：Successful biofilm secretion and expression</b></h4> | <h4><b>1. Congo Red：Successful biofilm secretion and expression</b></h4> | ||
| − | <b>His-CsgA-SpyCatcher-Histag</b><p></p> | + | <b class="tc">His-CsgA-SpyCatcher-Histag</b><p></p> |
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> | 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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| − | <b>His-CsgA-SpyCatcher</b><p></p> | + | <b class="tc">His-CsgA-SpyCatcher</b><p></p> |
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> | 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> | ||
<h4><b>2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials</b></h4> | <h4><b>2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials</b></h4> | ||
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| − | <b>His-CsgA-SpyCatcher-Histag</b><p></p> | + | <b class="bg">His-CsgA-SpyCatcher-Histag</b><p></p> |
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> | 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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<p style="text-align:center"><b>Fig 10.</b> Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.</p> | <p style="text-align:center"><b>Fig 10.</b> Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.</p> | ||
| − | <b>His-CsgA-SpyCatcher</b><p></p> | + | <b class="tc">His-CsgA-SpyCatcher</b><p></p> |
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> | 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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| − | <h3 >SpyTag and SpyCatcher [5]</h3> | + | <h3 class="bg" >SpyTag and SpyCatcher [5]</h3> |
<h4><b>Introduction and Motivation</b></h4> | <h4><b>Introduction and Motivation</b></h4> | ||
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> | 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> | ||
Revision as of 17:46, 18 October 2016
