Difference between revisions of "Part:BBa K1175006"
(→‎Effect of Temperature and pH on Enzyme Activity)
 
(6 intermediate revisions by 4 users not shown)
Line 43: Line 43:
 
</table>
 
</table>
 
</html>
 
</html>
 +
The two plates above are cellulose plates stained with congo red, clearing zones are visible around the colonies where bglS is breaking down the cellulose
 +
 +
 +
=Thinker-Shenzhen 2023=
 +
===Description===
 +
Considering the low degradation efficiency of alginate, we decided to construct cellulase Bgls, which will function with alginate lyase to break down alginate into much smaller molecules. It is derived from Bacillus Subtilis and is capable of depolymerizing cellulose, which can be tranferred into carbon source.
 +
===Usage and Biology===
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-12.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 1. The design of  bgls.</p >
 +
</div>
 +
</html>
 +
 +
We use T7 promoter to initiate the transcription and BBa_B0034 as RBS to start translation gene sequence of Endoglucanase, Exoglucanase, and β-glucosidase [1]. We choose BBa_B0015 as the terminator, which terminates the transcription. The Cellulase Bgls gene sequence is cloned into pET23b vector and expressed in E.coli Rosetta.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-13.png" style="width: 800px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 2. Mechanism of Cellulase</p >
 +
</div>
 +
</html>
 +
 +
===Characterization===
 +
To evaluate the expression and activity of Bacillus subtilis cellulase Bgls in E. coli Rosetta, we first synthesized the bgls gene and cloned it into the pET23b vector.  The recombinant vector was transformed into E. coli Rosetta cells and positive clones were screened on LB agar plates containing ampicillin.  Positive clones were verified by DNA sequencing.  Engineered E. coli Rosetta was cultured in LB broth medium for 3 days at 37°C, and then centrifuged at 13,000 rpm for 5 minutes.  Cell pellet was resuspended in PBS buffer (10 mM, pH 7.4) and then lysed through ultrasonication (150 W, 1s sonication with 3s intervals, for a total of 20 minutes).  1 ml of the supernatant (crude enzyme solution) was mixed with one ml of 1% carboxymethylcellulose (CMC, Sigma-Aldrich) solubilized in PBS buffer (10 mM, pH 7.4) and incubated at 37°C for 30 minutes under shaking (120 rpm).  One ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the mixture was boiled for 5 minutes, then the absorbance was measured at 540 nm.
 +
===1.Validation of Cellulase Bgls===
 +
The activity of the control group is 0.5 U/mg. Under the experimental conditions, which are 37℃ and pH 7.4, the specific enzyme activity of Cellulase Bgls crude enzyme mixture is 11.45 U/mg, which means that each milligram of crude enzyme mixture is capable of releasing 12 micromoles of reducing sugar per minute. Therefore, it proves the activity of Cellulase Bgls, indicating the result that Cellulase Bgls is functioning at relative efficient rate.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-14.png" style="width: 400px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 3. Th Graph of the Cellulase Activity of Control Group E.coli Rosetta and pT7-Bgls.</p >
 +
</div>
 +
</html>
 +
 +
Different concentrations of Bovine serum albumin (BSA) were conducted for plotting standard curve.  Cellulase specific activity was calculated by dividing the product concentration (μmol reducing sugars/min) expressed as units by the total protein (mg) of the sample.  All experiments were performed in triplicate, and the data are presented as mean values with SD.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-15.png" style="width: 400px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 4. Unpaired t Test of the Activity of Cellulase Bgls</p >
 +
</div>
 +
</html>
 +
 +
===2.The optimal reaction temperature for Cellulase Bgls===
 +
To determine the optimal reaction for Cellulase Bgls, we mixed the crude enzyme mixture with 1% CMC solution and incubated the mixture under 25℃, 37℃, and 55℃ for at least 30 minutes. After then, we used DNS (3, 5-dinitrosalicylic acid) test to find the concentration of the reducing sugar. As the graph shown, Cellulase Bgls performs the highest cellulase activity under 37 ℃.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-16.png" style="width: 400px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 5. Th Graph of the Cellulase Activity of Cellulase Bgls under three different temperatures.</p >
 +
</div>
 +
</html>
 +
 +
===3.The Optimal pH Value of reaction for Cellulase Bgls===
 +
To determine the optimal pH Value of reaction for Cellulase Bgls, we mixed the crude enzyme mixture with 1% CMC solution and incubated the mixture with the PBS buffer, which help to minimize the changes of pH value, at pH 5.8, pH 6.5, and pH 7.4 for at least 30 minutes. After the incubation, we used DNS (3, 5-dinitrosalicylic acid) test to determine the concentration of reducing sugar. Based on the graph below, the optimal pH value for Cellulase Bgls to perform reaction is 6.5.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/4979/wiki/part/basic-parts-2-bgls-old-part-contribution/image-17.png" style="width: 400px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 6. Th Graph of the Cellulase Activity of Cellulase Bgls at three different pH value.</p >
 +
</div>
 +
</html>
 +
 +
===Potential application directions===
 +
Under the experimental verification, cellulase Bgls is able to depolymerize cellulose, converting them into usable energy, which is an effcient and environmentally friendly way to develop energy. While cellulose is one of the most important components of plant cell wall and is abundant in the natural environment, the process of hydrolyzing cellulose by Cellulase Bgls has a wide range of applications in environmental protection (waste treatment), agriculture (fertilizer), bioengineering (bioethanol production), and industrial production (textiles and paper).
 +
===References===
 +
[1] Ejaz U, Sohail M, Ghanemi A. Cellulases: From Bioactivity to a Variety of Industrial Applications. Biomimetics (Basel). 2021 Jul 5;6(3):44. doi: 10.3390/biomimetics6030044. PMID: 34287227; PMCID: PMC8293267.
 +
 +
  
 
Copyright permission submitted
 
Copyright permission submitted
 +
 +
 +
=Contribution From GEC-Beijing=
 +
==Description==
 +
The aim of this experiment is to construct a cellulase-producing bacterial strain and evaluate the overexpression of cellulase in the engineered strain. Cellulase is widely used in the biostoning process for denim, where it breaks down cellulose fibers to create a worn or faded look on jeans. By optimizing cellulase production in the bacterial strain, we aim to improve the efficiency of denim processing and enhance fabric quality.
 +
==Usage and Biology==
 +
A cellulase-producing strain was constructed by integrating the cellulase gene Bgls (BBa_K1175006) from Bacillus subtilis into the pET23b plasmid, which was then transformed into Escherichia coli BL21 (Verification see Figure 2). The engineered strain, BL21/pET23b-Bgls, was inoculated in LB medium and incubated at 37℃ for 3 days.
 +
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/the-gene-circuit-of-bgls.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 1. The gene circuit of Bgls.</p >
 +
</div>
 +
</html>
 +
 +
==Characterization==
 +
===Cellulase Activity Assay===
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/gel-image-of-the-bgls-and-bupl.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 2. Gel image of the Bgls and Bpul. The Bpul gene is related to laccase and will be studied in later experiments.</p >
 +
</div>
 +
</html>
 +
 +
We used carboxymethyl cellulose (CMC) as the substrate to test the cellulase activity of the engineered strain. After sonicating the bacterial cells and collecting the crude enzyme solution, we measured the glucose produced from cellulose degradation. The results showed that the engineered strain BL21/pET23b-Bgls exhibited significantly higher cellulase activity compared to the empty vector control group.
 +
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/comparison-of-glucose-production-by-bl21-pet23b-bgls-and-bl21-pet23b-over-different-incubation-times.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 3. Comparison of glucose production by BL21/pET23b-Bgls and BL21/pET23b over different incubation times.</p >
 +
</div>
 +
</html>
 +
 +
The engineered strain BL21/p23b-Bgls demonstrated a much higher cellulase activity compared to the control strains, indicating its strong ability to degrade cellulose.
 +
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/cellulase-activity-of-engineered-bacterial-strains.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 4. Cellulase activity of engineered bacterial strains.</p >
 +
</div>
 +
</html>
 +
 +
===Effect of Temperature and pH on Enzyme Activity===
 +
We further tested the enzyme's activity under different temperatures and pH conditions. The results demonstrated that BL21/pET23b-Bgls shows the highest activity at 30℃ and pH=5.3, indicating that these are the optimal conditions for cellulose degradation.
 +
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/effect-of-temperature-on-cellulase-activity.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 5. Effect of temperature on cellulase activity.</p >
 +
</div>
 +
</html>
 +
 +
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5458/effect-of-ph-on-cellulase-activity.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 6. Effect of pH on cellulase activity.</p >
 +
</div>
 +
</html>
 +
 +
==Potential application directions==
 +
The discovery of optimal conditions for cellulase activity—30°C and pH=5.3—opens up new possibilities for industrial applications. These findings suggest that the engineered strain BL21/pET23b-Bgls can be utilized effectively in processes such as denim biostoning, where precise enzyme activity is crucial for controlled fabric treatment. Furthermore, the enzyme’s efficiency at moderate temperatures and acidic conditions makes it ideal for use in textile and paper industries, as well as in biofuel production, where the degradation of cellulose into glucose is essential. This environmentally friendly process could replace harsh chemical treatments, offering a sustainable alternative in various industries that require cellulose degradation.
 +
==References==
 +
Wood T M, Bhat K M. Methods for measuring cellulase activities[M]//Methods in enzymology. Academic Press, 1988, 160: 87-112.
 +
 +
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 04:03, 30 September 2024

endo-beta-1,3-1,4 glucanase (BglS) from Bacillus Subtilis 168

BglS This gene encodes an endo-beta-1,3-1,4-glucanase (bglS), which is from the bacterium Bacillus subtilis subtilis 168. The enzyme will hydrolyze and thereby cleave internal 1,4 linkages adjacent to 1,3 linkages.

Usage and Biology The substrates vulnerable to the bglS encoded enzyme are mixed linked beta-glucans. These glucans would have 1,3 and 1,4 beta linkages within the polysaccharide linking together the glucose monomers. Examples of these glucans can be found in oats, maize, and barley.

(1) http://mic.sgmjournals.org/content/141/2/281.long (2) http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1984.tb04259.x/pdf

bglS

The endo-1,3-1,4-glucanase bglS is a globular protein that that has two residues of interest. The putative nucleophile and acid-base cleavage sites at the E residues 133 and 137 highlighted in red.

bglS Enzyme Activity

Table 2. Substrate specificity of 1,3-1,4-beta-glucanase (BglS) purified to electrophoretic homogenity from E. coli cells harboring recombinant plasmid pRB33. Enzyme activities were calculated from the results of three independent measurements. *Unit defined as 1 micromole reducing sugar min-1 (mg purified enzyme)-1. **Unit defined as 1 OD595 unit min-1 (mg purified enzyme)-1. Table and data from: http://mic.sgmjournals.org/content/141/2/281.long The plates shown are differential medias containing both lichenan ,able to be broken down by the Bacillus Subtilis Subtilis 168 BglS gene. The plates were stained by flooding the plate with Congo red and the visualization of clearing zones was improved by flooding the stained plates with 1M NaCl. The Upper plate indicates, due to an apparent lack of clearing zones, that the strains MW14 (Deleted BglS), MW10 (Deleted EglS102 and Bgl BglSRV) and MW9 (Deletion of EglS and BglS) do not contain a significant ability to break down Lichenan. Whereas the strains MW8 (Deletion of EglS) and DB104 (No deletions) were able to break down the lichenan. The lower plate indicates, due to an apparent lack of clearing zones, that the strains MW10, MW8, and MW9 did not contain a significant ability to break down the CM-cellulose. On the other side of the coin, however, the strains MW14 and DB104 did have the ability to produce clearing zones, indicating their ability to break down CM-cellulose.

bglS on Cellulose Plates

The two plates above are cellulose plates stained with congo red, clearing zones are visible around the colonies where bglS is breaking down the cellulose


Thinker-Shenzhen 2023

Description

Considering the low degradation efficiency of alginate, we decided to construct cellulase Bgls, which will function with alginate lyase to break down alginate into much smaller molecules. It is derived from Bacillus Subtilis and is capable of depolymerizing cellulose, which can be tranferred into carbon source.

Usage and Biology

Figure 1. The design of bgls.

We use T7 promoter to initiate the transcription and BBa_B0034 as RBS to start translation gene sequence of Endoglucanase, Exoglucanase, and β-glucosidase [1]. We choose BBa_B0015 as the terminator, which terminates the transcription. The Cellulase Bgls gene sequence is cloned into pET23b vector and expressed in E.coli Rosetta.

Figure 2. Mechanism of Cellulase

Characterization

To evaluate the expression and activity of Bacillus subtilis cellulase Bgls in E. coli Rosetta, we first synthesized the bgls gene and cloned it into the pET23b vector. The recombinant vector was transformed into E. coli Rosetta cells and positive clones were screened on LB agar plates containing ampicillin. Positive clones were verified by DNA sequencing. Engineered E. coli Rosetta was cultured in LB broth medium for 3 days at 37°C, and then centrifuged at 13,000 rpm for 5 minutes. Cell pellet was resuspended in PBS buffer (10 mM, pH 7.4) and then lysed through ultrasonication (150 W, 1s sonication with 3s intervals, for a total of 20 minutes). 1 ml of the supernatant (crude enzyme solution) was mixed with one ml of 1% carboxymethylcellulose (CMC, Sigma-Aldrich) solubilized in PBS buffer (10 mM, pH 7.4) and incubated at 37°C for 30 minutes under shaking (120 rpm). One ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the mixture was boiled for 5 minutes, then the absorbance was measured at 540 nm.

1.Validation of Cellulase Bgls

The activity of the control group is 0.5 U/mg. Under the experimental conditions, which are 37℃ and pH 7.4, the specific enzyme activity of Cellulase Bgls crude enzyme mixture is 11.45 U/mg, which means that each milligram of crude enzyme mixture is capable of releasing 12 micromoles of reducing sugar per minute. Therefore, it proves the activity of Cellulase Bgls, indicating the result that Cellulase Bgls is functioning at relative efficient rate.

Figure 3. Th Graph of the Cellulase Activity of Control Group E.coli Rosetta and pT7-Bgls.

Different concentrations of Bovine serum albumin (BSA) were conducted for plotting standard curve. Cellulase specific activity was calculated by dividing the product concentration (μmol reducing sugars/min) expressed as units by the total protein (mg) of the sample. All experiments were performed in triplicate, and the data are presented as mean values with SD.

Figure 4. Unpaired t Test of the Activity of Cellulase Bgls

2.The optimal reaction temperature for Cellulase Bgls

To determine the optimal reaction for Cellulase Bgls, we mixed the crude enzyme mixture with 1% CMC solution and incubated the mixture under 25℃, 37℃, and 55℃ for at least 30 minutes. After then, we used DNS (3, 5-dinitrosalicylic acid) test to find the concentration of the reducing sugar. As the graph shown, Cellulase Bgls performs the highest cellulase activity under 37 ℃.

Figure 5. Th Graph of the Cellulase Activity of Cellulase Bgls under three different temperatures.

3.The Optimal pH Value of reaction for Cellulase Bgls

To determine the optimal pH Value of reaction for Cellulase Bgls, we mixed the crude enzyme mixture with 1% CMC solution and incubated the mixture with the PBS buffer, which help to minimize the changes of pH value, at pH 5.8, pH 6.5, and pH 7.4 for at least 30 minutes. After the incubation, we used DNS (3, 5-dinitrosalicylic acid) test to determine the concentration of reducing sugar. Based on the graph below, the optimal pH value for Cellulase Bgls to perform reaction is 6.5.

Figure 6. Th Graph of the Cellulase Activity of Cellulase Bgls at three different pH value.

Potential application directions

Under the experimental verification, cellulase Bgls is able to depolymerize cellulose, converting them into usable energy, which is an effcient and environmentally friendly way to develop energy. While cellulose is one of the most important components of plant cell wall and is abundant in the natural environment, the process of hydrolyzing cellulose by Cellulase Bgls has a wide range of applications in environmental protection (waste treatment), agriculture (fertilizer), bioengineering (bioethanol production), and industrial production (textiles and paper).

References

[1] Ejaz U, Sohail M, Ghanemi A. Cellulases: From Bioactivity to a Variety of Industrial Applications. Biomimetics (Basel). 2021 Jul 5;6(3):44. doi: 10.3390/biomimetics6030044. PMID: 34287227; PMCID: PMC8293267.


Copyright permission submitted


Contribution From GEC-Beijing

Description

The aim of this experiment is to construct a cellulase-producing bacterial strain and evaluate the overexpression of cellulase in the engineered strain. Cellulase is widely used in the biostoning process for denim, where it breaks down cellulose fibers to create a worn or faded look on jeans. By optimizing cellulase production in the bacterial strain, we aim to improve the efficiency of denim processing and enhance fabric quality.

Usage and Biology

A cellulase-producing strain was constructed by integrating the cellulase gene Bgls (BBa_K1175006) from Bacillus subtilis into the pET23b plasmid, which was then transformed into Escherichia coli BL21 (Verification see Figure 2). The engineered strain, BL21/pET23b-Bgls, was inoculated in LB medium and incubated at 37℃ for 3 days.

Figure 1. The gene circuit of Bgls.

Characterization

Cellulase Activity Assay

Figure 2. Gel image of the Bgls and Bpul. The Bpul gene is related to laccase and will be studied in later experiments.

We used carboxymethyl cellulose (CMC) as the substrate to test the cellulase activity of the engineered strain. After sonicating the bacterial cells and collecting the crude enzyme solution, we measured the glucose produced from cellulose degradation. The results showed that the engineered strain BL21/pET23b-Bgls exhibited significantly higher cellulase activity compared to the empty vector control group.

Figure 3. Comparison of glucose production by BL21/pET23b-Bgls and BL21/pET23b over different incubation times.

The engineered strain BL21/p23b-Bgls demonstrated a much higher cellulase activity compared to the control strains, indicating its strong ability to degrade cellulose.

Figure 4. Cellulase activity of engineered bacterial strains.

Effect of Temperature and pH on Enzyme Activity

We further tested the enzyme's activity under different temperatures and pH conditions. The results demonstrated that BL21/pET23b-Bgls shows the highest activity at 30℃ and pH=5.3, indicating that these are the optimal conditions for cellulose degradation.

Figure 5. Effect of temperature on cellulase activity.


Figure 6. Effect of pH on cellulase activity.

Potential application directions

The discovery of optimal conditions for cellulase activity—30°C and pH=5.3—opens up new possibilities for industrial applications. These findings suggest that the engineered strain BL21/pET23b-Bgls can be utilized effectively in processes such as denim biostoning, where precise enzyme activity is crucial for controlled fabric treatment. Furthermore, the enzyme’s efficiency at moderate temperatures and acidic conditions makes it ideal for use in textile and paper industries, as well as in biofuel production, where the degradation of cellulose into glucose is essential. This environmentally friendly process could replace harsh chemical treatments, offering a sustainable alternative in various industries that require cellulose degradation.

References

Wood T M, Bhat K M. Methods for measuring cellulase activities[M]//Methods in enzymology. Academic Press, 1988, 160: 87-112.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]