Difference between revisions of "Part:BBa K2564000"

(→‎Contribution)
 
(7 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2564000 short</partinfo>
 
<partinfo>BBa_K2564000 short</partinfo>
  
−
The molecular function of beta-glucosidase (bgl1A) is Catalysis of the hydrolysis of terminal, non-reducing beta-D-glucose residues with release of beta-D-glucose.
+
The molecular function of Beta-glucosidase (Bgl1A) is Catalysis of the hydrolysis of terminal, non-reducing Beta-D-glucose residues with release of Beta-D-glucose.
  
−
==Contribution==
+
==Contribution ==
−
'''Group''': [http://2019.igem.org/Team:XMU-China iGEM Team XMU-China 2019]
+
'''Group''': [http://2019.igem.org/Team/XMU-China iGEM Team XMU-China 2019]
  
 
'''Author''': Jisheng Xie, Zinuo Huang
 
'''Author''': Jisheng Xie, Zinuo Huang
Line 15: Line 15:
 
'''Bgl1A'''
 
'''Bgl1A'''
  
−
Cellulose is a polymer composed of beta-1,4-linked glucosyl residues. Cellulases (Endoglucanases), cellobiosidases (Exoglucanases), and Beta-glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.  
+
Cellulose is a polymer composed of Beta-1,4-linked glucosyl residues. Cellulases (Endoglucanases), Cellobiosidases (Exoglucanases), and Beta-glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.  
  
−
Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in beta-D-glucosides and oligosaccharides, with release of glucose.<ref>M. Cox, D. Nelson, Lehninger Principles of Biochemistry.  (2000), vol. 5. New York: Worth Publishers. pp. 306–308.</ref>
+
Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in Beta-D-glucosides and oligosaccharides, with release of glucose.<ref>M. Cox, D. Nelson, Lehninger Principles of Biochemistry.  (2000), vol. 5. New York: Worth Publishers. pp. 306–308.</ref>
  
−
Depending on the organism cellobiose may be cleaved extracellularly by β-glucosidases (cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.<ref>R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).</ref>
+
Depending on the organism cellobiose may be cleaved extracellularly by Beta-glucosidases (Cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.<ref>R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).</ref>
  
  
Line 27: Line 27:
  
  
−
====Enzyme digestion====
+
====Restriction digestion====
 
We verified it by restriction digestion before using it.
 
We verified it by restriction digestion before using it.
 
<html>
 
<html>
Line 39: Line 39:
 
     </figure>
 
     </figure>
 
</html>
 
</html>
−
:'''Fig. 1''' Agarose Gel Electrophoresis of T7-RBS-bgl1A (BBa_K2564000). (M: Marker)
+
:'''Fig. 1''' Agarose Gel Electrophoresis of T7-RBS-bgl1A (<partinfo>BBa_K2564000</partinfo>). (M: Marker)
 +
 
 +
The part was insert into the expression vectors with T7 and RBS (<partinfo>BBa_K525998</partinfo>). Then transformed the expression vectors into ''E. coli'' DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.
 +
 
 +
<html>
 +
    <figure>
 +
        <img src="https://2019.igem.org/wiki/images/6/65/T--XMU-China--bgl1A.png" height="70" style="float:center">
 +
        <br>
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 
 +
====SDS-PAGE====
 +
We transformed the constructed plasmid into ''E. coli'' BL21 (DE3). The positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/ol) polyacrylamide gel, followed by Coomassie blue staining. (Fig. 2)
 +
 
 +
<html>
 +
    <figure>
 +
        <img src="https://2019.igem.org/wiki/images/8/82/T--XMU-China--T7-RBS-bgl1A_SDS-PAGE.png" height="300" style="float:center">
 +
        <br>
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
:'''Fig. 2''' SDS-PAGE analysis of protein in ''E. coli'' BL21 (DE3) cells and the medium by Coomassie blue staining. Bgl1A: protein of BL21 (DE3) carrying T7-RBS-bgl1A (linked by BBa_K525998 and BBa_K2564000); Control: protein of BL21 (DE3) carrying T7 and RBS (BBa_K525998).
  
  
 
====HPLC Quantitative Experiment====
 
====HPLC Quantitative Experiment====
−
We use HPLC to verify the activity of bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.  
+
We use HPLC to verify the activity of Bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.  
  
 
<html>
 
<html>
Line 55: Line 83:
 
     </figure>
 
     </figure>
 
</html>
 
</html>
−
:'''Fig. 2''' SWC for T7-RBS-bgl1A, made through the relationship between peak area and concentration.
+
:'''Fig. 3''' SWC for T7-RBS-bgl1A, made through the relationship between peak area and concentration.
  
  
Line 70: Line 98:
 
     </figure>
 
     </figure>
 
</html>
 
</html>
−
:'''Fig. 3''' The results of HPLC. (A): T7-RBS-bgl1A supernatant; (B): T7-RBS-bgl1A broken supernatant  
+
:'''Fig. 4''' The results of HPLC. (A): T7-RBS-bgl1A supernatant; (B): T7-RBS-bgl1A broken supernatant.
  
−
Result of the broken supernatant of medium cultures with PT7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained, which means that Bgl1A can degrade D-cellobiose into D-glucose.
+
Result of the broken supernatant of medium cultures with T7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained, which means that Bgl1A can degrade D-cellobiose into D-glucose.
  
−
Supernatant of medium cultures with PT7-RBS-bgl1A part shows that D-cellobiose didn't get consumed with extension of reaction time and D-glucose didn't increase, which means that Bgl1A didn't secret out into the medium.
+
Supernatant of medium cultures with T7-RBS-bgl1A part shows that D-cellobiose didn't get consumed with extension of reaction time and D-glucose didn't increase, which means that Bgl1A secreted out into the medium don't have enzymatic activity.
  
  

Latest revision as of 21:27, 21 October 2019


Beta-glucosidase (bgl1A)

The molecular function of Beta-glucosidase (Bgl1A) is Catalysis of the hydrolysis of terminal, non-reducing Beta-D-glucose residues with release of Beta-D-glucose.

Contribution

Group: [http://2019.igem.org/Team/XMU-China iGEM Team XMU-China 2019]

Author: Jisheng Xie, Zinuo Huang

Summary: Enzyme digestion and enzyme activity assay

Biology

Bgl1A

Cellulose is a polymer composed of Beta-1,4-linked glucosyl residues. Cellulases (Endoglucanases), Cellobiosidases (Exoglucanases), and Beta-glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in Beta-D-glucosides and oligosaccharides, with release of glucose.[1]

Depending on the organism cellobiose may be cleaved extracellularly by Beta-glucosidases (Cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.[2]


Characterization from iGEM19-XMU-China

Molecular weight

This gene codes for a protein of 461 amino acids with a molecular mass of 52,754 Da.


Restriction digestion

We verified it by restriction digestion before using it.


Fig. 1 Agarose Gel Electrophoresis of T7-RBS-bgl1A (BBa_K2564000). (M: Marker)

The part was insert into the expression vectors with T7 and RBS (BBa_K525998). Then transformed the expression vectors into E. coli DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.


SDS-PAGE

We transformed the constructed plasmid into E. coli BL21 (DE3). The positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/ol) polyacrylamide gel, followed by Coomassie blue staining. (Fig. 2)


Fig. 2 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Bgl1A: protein of BL21 (DE3) carrying T7-RBS-bgl1A (linked by BBa_K525998 and BBa_K2564000); Control: protein of BL21 (DE3) carrying T7 and RBS (BBa_K525998).


HPLC Quantitative Experiment

We use HPLC to verify the activity of Bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.


Fig. 3 SWC for T7-RBS-bgl1A, made through the relationship between peak area and concentration.


Then mix the crude enzyme solution with cellobiose, incubate under the condition of 37°C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction when and after interval time since reaction started. And then carry out HPLC on the sample.


Fig. 4 The results of HPLC. (A): T7-RBS-bgl1A supernatant; (B): T7-RBS-bgl1A broken supernatant.

Result of the broken supernatant of medium cultures with T7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained, which means that Bgl1A can degrade D-cellobiose into D-glucose.

Supernatant of medium cultures with T7-RBS-bgl1A part shows that D-cellobiose didn't get consumed with extension of reaction time and D-glucose didn't increase, which means that Bgl1A secreted out into the medium don't have enzymatic activity.


Reference

  1. ↑ M. Cox, D. Nelson, Lehninger Principles of Biochemistry. (2000), vol. 5. New York: Worth Publishers. pp. 306–308.
  2. ↑ R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1213
  • 1000
    COMPATIBLE WITH RFC[1000]