Difference between revisions of "Part:BBa K2711000"
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The UiOslo_Norway 2018 team used the sequence of endo-1,3-β-glucanase from Cellulosimicrobium cellulans (= Arthrobacter Luteus) to enable targeted lysis of yeast cells. This glucanase is also used in the commercially available product Zymolyase, among other components, which is a lytic enzyme for the digestion of yeast cell wall glucan. In our case we wanted to specifically lyse Candida albicans cells in a vaginal sample which will also contain for example bacteria and skin cells. The glucanase was synthesized by IDT after the sequence was codon optimized. | The UiOslo_Norway 2018 team used the sequence of endo-1,3-β-glucanase from Cellulosimicrobium cellulans (= Arthrobacter Luteus) to enable targeted lysis of yeast cells. This glucanase is also used in the commercially available product Zymolyase, among other components, which is a lytic enzyme for the digestion of yeast cell wall glucan. In our case we wanted to specifically lyse Candida albicans cells in a vaginal sample which will also contain for example bacteria and skin cells. The glucanase was synthesized by IDT after the sequence was codon optimized. | ||
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<h2>Endo-1,3-β-Glucanase</h2> | <h2>Endo-1,3-β-Glucanase</h2> | ||
<p>The enzyme glucanase is an important part of our detection system, as it breaks down the glucans in the yeast cell wall, making it easier to lyse the cell and access the DNA. We were not only able to successfully clone the endo-1,3-β-gluanase into the submission vector (Figure 1) and expression vector (Figure 2), but we were also able it induce its expression (Figure 3). Finally, we could also show the glucanase activity in vitro (Figure 4, Figure 5).</p> | <p>The enzyme glucanase is an important part of our detection system, as it breaks down the glucans in the yeast cell wall, making it easier to lyse the cell and access the DNA. We were not only able to successfully clone the endo-1,3-β-gluanase into the submission vector (Figure 1) and expression vector (Figure 2), but we were also able it induce its expression (Figure 3). Finally, we could also show the glucanase activity in vitro (Figure 4, Figure 5).</p> | ||
| − | + | <img class="imgshadow front" src="https://static.igem.org/mediawiki/2018/4/4d/T--UiOslo_Norway--RFig1.png"> | |
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<p><strong>Figure 1: Colony PCR of glucanase in submission vector pSB1C3</strong></br>Positive colonies after Two-part Gibson transformation of glucanase g-block (1-15). Glucanase at the size of 1.388 bp (blue line), colony 11 was further used after checking for correct sequence. Ladder Quick-Load 1 kb (N0468S).</p> | <p><strong>Figure 1: Colony PCR of glucanase in submission vector pSB1C3</strong></br>Positive colonies after Two-part Gibson transformation of glucanase g-block (1-15). Glucanase at the size of 1.388 bp (blue line), colony 11 was further used after checking for correct sequence. Ladder Quick-Load 1 kb (N0468S).</p> | ||
| − | + | <img class="imgshadow front" src="https://static.igem.org/mediawiki/2018/f/f6/T--UiOslo_Norway--RFig2.png"> | |
<p><strong>Figure 2: Colony PCR of glucanase in expression vector pBAD</strong></br>Positive colonies after Two-part Gibson transformation of glucanase (1-9). Glucanase at the size of 1.388 bp plus overhangs (blue line). Ladder Quick-Load 1 kb (N0468S).</p> | <p><strong>Figure 2: Colony PCR of glucanase in expression vector pBAD</strong></br>Positive colonies after Two-part Gibson transformation of glucanase (1-9). Glucanase at the size of 1.388 bp plus overhangs (blue line). Ladder Quick-Load 1 kb (N0468S).</p> | ||
<p>The glucanase-pBAD construct was further used for protein expression. Protein expression was induced by adding arabinose (protocols).</p> | <p>The glucanase-pBAD construct was further used for protein expression. Protein expression was induced by adding arabinose (protocols).</p> | ||
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<h2>Induction of protein expression</h2> | <h2>Induction of protein expression</h2> | ||
<p>The induction of protein expression was verified after small-/or medium-scale cell culturing (protocol),followined by separation of proteins on a 12 % SDS-PAGE (Figure 3). The results show that the endo-1,3-β -glucanase expression was induced after addition of 0.1 % arabinose followed by a 3 h incubation at 30<sup>o</sup>C (protocol).</p> | <p>The induction of protein expression was verified after small-/or medium-scale cell culturing (protocol),followined by separation of proteins on a 12 % SDS-PAGE (Figure 3). The results show that the endo-1,3-β -glucanase expression was induced after addition of 0.1 % arabinose followed by a 3 h incubation at 30<sup>o</sup>C (protocol).</p> | ||
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| + | <img style="width:40% !important;"class="imgshadow front" src="https://static.igem.org/mediawiki/2018/3/32/T--UiOslo_Norway--RFig3.png"> | ||
<p><strong>Figure 3: SDS-PAGE 12 %</strong></br>Uninduced (1) and induced samples (2&3) loaded on SDS-PAGE. Glucanase at the size of 52 kDa (red arrow). | <p><strong>Figure 3: SDS-PAGE 12 %</strong></br>Uninduced (1) and induced samples (2&3) loaded on SDS-PAGE. Glucanase at the size of 52 kDa (red arrow). | ||
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<p>Insoluble dyed substrate is used to detect enzyme activity (protocol).</p> | <p>Insoluble dyed substrate is used to detect enzyme activity (protocol).</p> | ||
| − | + | <img class="imgshadow front" src="https://static.igem.org/mediawiki/2018/5/5a/T--UiOslo_Norway--RFig6.png"> | |
<p><strong>Figure 4: 96-well plate assay to test enzyme activity (Megazyme)</strong></br> | <p><strong>Figure 4: 96-well plate assay to test enzyme activity (Megazyme)</strong></br> | ||
Absorbance of the glucanase was measured against diluted samples of a standard for enzyme activity (Zymolyase, Nordic Biosite). The glucanase activity was tested with samples from induced cell cultures (crude extract, supernatant and cell pellet). Absorbance was measured 1 h after loading the samples on the 96-well plate. </p> | Absorbance of the glucanase was measured against diluted samples of a standard for enzyme activity (Zymolyase, Nordic Biosite). The glucanase activity was tested with samples from induced cell cultures (crude extract, supernatant and cell pellet). Absorbance was measured 1 h after loading the samples on the 96-well plate. </p> | ||
<p>From Figure 4, it can be seen that the glucanase had an activity in between a 100x and 1.000x dilution of Zymolyase. Of the different cell samples from the induced cell culture, the glucanase from the supernatant had the highest enzymatic activity. We expect the glucanase to have a higher enzymatic activity after further experiments purifying the protein.</p> | <p>From Figure 4, it can be seen that the glucanase had an activity in between a 100x and 1.000x dilution of Zymolyase. Of the different cell samples from the induced cell culture, the glucanase from the supernatant had the highest enzymatic activity. We expect the glucanase to have a higher enzymatic activity after further experiments purifying the protein.</p> | ||
| − | + | <img style="width:40%;" src="https://static.igem.org/mediawiki/2018/5/58/T--UiOslo_Norway--RFig5.png"> | |
<p><strong>Figure 5: Plate assay to display enzyme activity (Glycospot)</strong> </br>Three replicates of assay. Upper left corner ‘crude cell culture’, middle left ‘supernatant’, lower left ‘positive control’ (commercially available enzyme mix), upper right ‘crude diluted’, middle right ‘pellet’, lower right ‘negative control’ (empty pBAD vector). All samples on the left parts of the plates show enzyme activity in all replicates. </p> | <p><strong>Figure 5: Plate assay to display enzyme activity (Glycospot)</strong> </br>Three replicates of assay. Upper left corner ‘crude cell culture’, middle left ‘supernatant’, lower left ‘positive control’ (commercially available enzyme mix), upper right ‘crude diluted’, middle right ‘pellet’, lower right ‘negative control’ (empty pBAD vector). All samples on the left parts of the plates show enzyme activity in all replicates. </p> | ||
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| − | SOURCES: | + | </h2>SOURCES:</h2> |
1. Deepak Mudgil, in Dietary Fiber for the Prevention of Cardiovascular Disease, 2017 | 1. Deepak Mudgil, in Dietary Fiber for the Prevention of Cardiovascular Disease, 2017 | ||
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2. Salazar et al., 2001. Overproduction, Purification, and Characterization of β-1,3- Glucanase Type II in Escherichia coli. Protein Expression and Purification 23, 219–225. | 2. Salazar et al., 2001. Overproduction, Purification, and Characterization of β-1,3- Glucanase Type II in Escherichia coli. Protein Expression and Purification 23, 219–225. | ||
| − | 3. Oda et al., 2017. Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism. BBA - Proteins and Proteomics 1866 (2018) 415–425. | + | 3. Oda et al., 2017. Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism. BBA - Proteins and Proteomics 1866 (2018) 415–425.<br><br> |
| + | <b>Improvement from TARTU_TUIT2019</b><br><br> | ||
| + | One part of our project was connected to the improvement of a previously described part BBa_K2711000 added to the registry by UiOslo_Norway 2018 team. BBa_K2711000 (BBa) is a bacterial gene from <i>Arthrobacter luteus</i> coding for glucan hydrolase. As our goal was to induce yeast self-lysis, we needed to improve the functioning of BBa_K2711000 when expressed in yeast. As the first step, we codon-optimized it for use in <i>Saccharomyces cerevisiae</i>. BBa_K2711000 protein is part of a commercial enzyme mixture Zymolyase commonly used for yeast cell wall dissolution. When expressed in bacteria, BBa_K2711000 is secreted from the cells due to the presence of Tat-type secretion signal. This type of secretion signal, however, is inactive in yeast, so presumably, when expressed in yeast, BBa_K2711000 will accumulate in the cytoplasm, where it cannot affect the cell wall. To prevent it from accumulating in the cell and to increase its concentration close to the cell wall we have added yeast secretion signals from α-factor and Ost1 to the N-terminus of BBa_K2711000.<br><br> | ||
| + | We tested the activity of different BBa constructs using time-lapse microscopy to follow the effect of BBa expression induced at the start of the experiment. In the microscopy experiments it can be seen that while expression of BBa_K2711000 does have an effect on cell viability, the effect is more pronounced if BBa is fused with eukaryotic secretion signals. From the movies, induced lysis of some cells can be seen in case of Ost1-BBa expression, confirming that BBa_K2711000 when fused with Ost1 secretion signal, is able to promote yeast self-lysis. Additionally, 13% of cells expressing Ost1-BBa were dead after 20 hours of induction, compared to 7% of cells expressing unmodified BBa_K2711000 (Fig. 6).<br> | ||
| + | Therefore, our team has contributed to the improvement and development of this biobrick for use in yeasts. <br><br> | ||
| + | Video 1 (https://2019.igem.org/wiki/images/b/b1/T--Tartu_TUIT--Ost1-BBa.mp4) time-lapse microscopy. On the right side, there are induced Ost1-BBa_K2711000 expressing cells. The left side depicts uninduced Ost1-BBa_K2711000 expressing cells. Yellow arrows indicate cell lysis. | ||
| + | </html> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 01:35, 22 October 2019
Glucanase
β-Glucan is a water-soluble polysaccharide that consists of glucose units. In yeast cell walls, glucose monomers are linked in a branched structure via β-(1→3) and β-(1→6) glycosidic bonds [1]. β-1,3-Glucanases (EC 3.2.1.39 and EC 3.2.1.6) catalyze the hydrolysis of glucans containing β-1,3-linked glucose monomers, one of the most abundant polysaccharides present in the yeast cell wall [2]. Bacterial endo-1,3-β-glucanases catalyze the hydrolysis of (13)-beta-D-glucosidic linkages in (1 → 3)-beta-D-glucans. These enzymes have a β-sandwich architecture and belong to the glycoside hydrolase family 16 (GH16) [3].
The UiOslo_Norway 2018 team used the sequence of endo-1,3-β-glucanase from Cellulosimicrobium cellulans (= Arthrobacter Luteus) to enable targeted lysis of yeast cells. This glucanase is also used in the commercially available product Zymolyase, among other components, which is a lytic enzyme for the digestion of yeast cell wall glucan. In our case we wanted to specifically lyse Candida albicans cells in a vaginal sample which will also contain for example bacteria and skin cells. The glucanase was synthesized by IDT after the sequence was codon optimized.
Endo-1,3-β-Glucanase
The enzyme glucanase is an important part of our detection system, as it breaks down the glucans in the yeast cell wall, making it easier to lyse the cell and access the DNA. We were not only able to successfully clone the endo-1,3-β-gluanase into the submission vector (Figure 1) and expression vector (Figure 2), but we were also able it induce its expression (Figure 3). Finally, we could also show the glucanase activity in vitro (Figure 4, Figure 5).
Figure 1: Colony PCR of glucanase in submission vector pSB1C3Positive colonies after Two-part Gibson transformation of glucanase g-block (1-15). Glucanase at the size of 1.388 bp (blue line), colony 11 was further used after checking for correct sequence. Ladder Quick-Load 1 kb (N0468S).
Figure 2: Colony PCR of glucanase in expression vector pBADPositive colonies after Two-part Gibson transformation of glucanase (1-9). Glucanase at the size of 1.388 bp plus overhangs (blue line). Ladder Quick-Load 1 kb (N0468S).
The glucanase-pBAD construct was further used for protein expression. Protein expression was induced by adding arabinose (protocols).
Induction of protein expression
The induction of protein expression was verified after small-/or medium-scale cell culturing (protocol),followined by separation of proteins on a 12 % SDS-PAGE (Figure 3). The results show that the endo-1,3-β -glucanase expression was induced after addition of 0.1 % arabinose followed by a 3 h incubation at 30oC (protocol).
Figure 3: SDS-PAGE 12 %Uninduced (1) and induced samples (2&3) loaded on SDS-PAGE. Glucanase at the size of 52 kDa (red arrow).
Screening for Polysaccharide Endo-Hydrolases using Insoluble Dyed Polysaccharide
Insoluble dyed substrate is used to detect enzyme activity (protocol).
Figure 4: 96-well plate assay to test enzyme activity (Megazyme) Absorbance of the glucanase was measured against diluted samples of a standard for enzyme activity (Zymolyase, Nordic Biosite). The glucanase activity was tested with samples from induced cell cultures (crude extract, supernatant and cell pellet). Absorbance was measured 1 h after loading the samples on the 96-well plate.
From Figure 4, it can be seen that the glucanase had an activity in between a 100x and 1.000x dilution of Zymolyase. Of the different cell samples from the induced cell culture, the glucanase from the supernatant had the highest enzymatic activity. We expect the glucanase to have a higher enzymatic activity after further experiments purifying the protein.
Figure 5: Plate assay to display enzyme activity (Glycospot) Three replicates of assay. Upper left corner ‘crude cell culture’, middle left ‘supernatant’, lower left ‘positive control’ (commercially available enzyme mix), upper right ‘crude diluted’, middle right ‘pellet’, lower right ‘negative control’ (empty pBAD vector). All samples on the left parts of the plates show enzyme activity in all replicates.
Conclusion
With our results we showed that our BioBrick, the endo- 1,3-β-glucanase, has enzymatic activity and is able to cleave 1,3-β-glucan bonds. The activity is comparable to a 100x-1000x dilution of Zymolyase. Combined with either mechanical force or a detergent, we expect to be able to selectively lyse yeast cell walls.
SOURCES: 1. Deepak Mudgil, in Dietary Fiber for the Prevention of Cardiovascular Disease, 2017 2. Salazar et al., 2001. Overproduction, Purification, and Characterization of β-1,3- Glucanase Type II in Escherichia coli. Protein Expression and Purification 23, 219–225. 3. Oda et al., 2017. Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism. BBA - Proteins and Proteomics 1866 (2018) 415–425.Improvement from TARTU_TUIT2019
One part of our project was connected to the improvement of a previously described part BBa_K2711000 added to the registry by UiOslo_Norway 2018 team. BBa_K2711000 (BBa) is a bacterial gene from Arthrobacter luteus coding for glucan hydrolase. As our goal was to induce yeast self-lysis, we needed to improve the functioning of BBa_K2711000 when expressed in yeast. As the first step, we codon-optimized it for use in Saccharomyces cerevisiae. BBa_K2711000 protein is part of a commercial enzyme mixture Zymolyase commonly used for yeast cell wall dissolution. When expressed in bacteria, BBa_K2711000 is secreted from the cells due to the presence of Tat-type secretion signal. This type of secretion signal, however, is inactive in yeast, so presumably, when expressed in yeast, BBa_K2711000 will accumulate in the cytoplasm, where it cannot affect the cell wall. To prevent it from accumulating in the cell and to increase its concentration close to the cell wall we have added yeast secretion signals from α-factor and Ost1 to the N-terminus of BBa_K2711000.
We tested the activity of different BBa constructs using time-lapse microscopy to follow the effect of BBa expression induced at the start of the experiment. In the microscopy experiments it can be seen that while expression of BBa_K2711000 does have an effect on cell viability, the effect is more pronounced if BBa is fused with eukaryotic secretion signals. From the movies, induced lysis of some cells can be seen in case of Ost1-BBa expression, confirming that BBa_K2711000 when fused with Ost1 secretion signal, is able to promote yeast self-lysis. Additionally, 13% of cells expressing Ost1-BBa were dead after 20 hours of induction, compared to 7% of cells expressing unmodified BBa_K2711000 (Fig. 6).
Therefore, our team has contributed to the improvement and development of this biobrick for use in yeasts.
Video 1 (https://2019.igem.org/wiki/images/b/b1/T--Tartu_TUIT--Ost1-BBa.mp4) time-lapse microscopy. On the right side, there are induced Ost1-BBa_K2711000 expressing cells. The left side depicts uninduced Ost1-BBa_K2711000 expressing cells. Yellow arrows indicate cell lysis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 958
- 1000COMPATIBLE WITH RFC[1000]