Difference between revisions of "Part:BBa K118023"

(→‎Congo Red Assay)
Line 11: Line 11:
 
<partinfo>BBa_K118023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K118023 SequenceAndFeatures</partinfo>
  
−
==Characterization from iGEM18-UESTC-China==
+
==Contribution ==
−
===Molecular weight===
+
*'''Group:''' [http://2018.igem.org/Team:UESTC-China iGEM Team UESTC-China 2018]
 +
*'''Author:''' Liang Zhao, Yetao Zou
 +
*'''Summary:''' CMC enzyme activity assay and congo red assay for enzyme activity
 +
 
 +
===Characterization from iGEM18-UESTC-China===
 +
====Molecular weight====
 
This gene codes for a protein of 448 amino acids with a molecular mass of 46.7 kDa.
 
This gene codes for a protein of 448 amino acids with a molecular mass of 46.7 kDa.
−
===Congo red assay===
+
====Congo Red Assay====
 
This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed.  
 
This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed.  
−
Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation. The results are shown in Fig.1.
+
Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24 h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1 h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation [1]. The results are shown in Fig. 1.
−
[[File:T--UESTC-China--Congo Red Assay.png|500px|thumb|center|'''Fig. 1''' Congo red assay. Line 1: BL21(DE3) carrying cenA gene. Line 2: Positive control enzyme.  Line 3: BL21(DE3) carrying empty vector control.]]
+
[[File:T--UESTC-China--Congo Red Assay.png|500px|thumb|center|'''Fig. 1''' Congo red assay. Line 1: BL21(DE3) carrying cenA gene. Line 2: Positive control enzyme.  Line 3: BL21(DE3) carrying empty vector control]]
−
The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC showed that cenA gene was successfully transcribed and translated by BL21(DE3). The empty vector control didn't show any zone of clearance around the colonies.
+
The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC. The empty vector control didn't show any zone of clearance around the colonies. The results showed that cenA gene can successfully works in BL21(DE3).
 +
 
 +
==== CMCase activity assay ====
 +
In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for endoglucanase activity. 
 +
[[File:T--UESTC-China-- CMCase activity assay.png|500px|thumb|center|'''Fig. 2''' endoglucanase activity assay]]
 +
As shown in Fig. 2, BL21(DE3) carrying cenA gene could decompose CMC-Na while negative control couldn't, which proved that cenA could work successfully.
  
−
===3,5-dinitrosalicylic acid (DNS) method ===
+
====References====
−
In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for cenA activity.Firstly, for quantitative assay, standard curves of ranging from 0-1000mg/L glucose were measured (Fig. 2).
+
−
[[File:T--UESTC-China--Standard Curve of Glucose.png|500px|thumb|center|'''Fig. 2''' Standard curve of glucose.]]
+
−
+
−
Reactions were carried out in 50ml centrifuge tubes with 1.0ml CMC-Na solution, 0.5ml crude enzyme solution and 0.5ml potassium phosphate buffer (50mM, pH 7.0). Reaction mixtures were incubated at 40 °C for 3 h, enzyme action was interrupted by the addition of 3,5-dinitrosalisylic acid (DNS) reagent, which was used to quantify the total amount of reducing sugars. Reaction mixtures were then placed in a boiling water bath for 5 min, cooled to room temperature and diluted to 25 ml with water for the measurement of absorbance at 540 nm with a spectrophotometer.
+
−
As shown in Fig. 3, BL21(DE3) carrying cenA gene could decompose CMC-Na while wild-type couldn't, which proved that cenA could work successfully.
+
  
  

Revision as of 08:42, 17 October 2018

cenA coding sequence encoding Cellulomonas fimi endoglucanase A

The cellulolytic bacterium Cellulomonas fimi uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 34
    Illegal NotI site found at 1178
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1091
    Illegal BamHI site found at 227
    Illegal XhoI site found at 589
    Illegal XhoI site found at 838
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 369
    Illegal NgoMIV site found at 1294
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 273

Contribution

  • Group: [http://2018.igem.org/Team:UESTC-China iGEM Team UESTC-China 2018]
  • Author: Liang Zhao, Yetao Zou
  • Summary: CMC enzyme activity assay and congo red assay for enzyme activity

Characterization from iGEM18-UESTC-China

Molecular weight

This gene codes for a protein of 448 amino acids with a molecular mass of 46.7 kDa.

Congo Red Assay

This year in order to find out if cenA gene had been expressed successfully in BL21(DE3), the method of Congo Red assay was performed. Luria agar plates with 0.2% CMC were inoculated with BL21(DE3) carrying cenA gene and incubated at 37°C for 24h. After 24 h the strain was scraped off. The agar was flooded with 1 mg/ml Congo Red solution for 1h. Congo Red solution was poured off into a toxic waste bottle and 1 M NaCl was added and left for another 1 h. Then NaCl solution was poured off. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation [1]. The results are shown in Fig. 1.

Fig. 1 Congo red assay. Line 1: BL21(DE3) carrying cenA gene. Line 2: Positive control enzyme. Line 3: BL21(DE3) carrying empty vector control

The strains carrying cenA showed a zone of clearance created by hydrolysis of CMC. The empty vector control didn't show any zone of clearance around the colonies. The results showed that cenA gene can successfully works in BL21(DE3).

CMCase activity assay

In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for endoglucanase activity.

Fig. 2 endoglucanase activity assay

As shown in Fig. 2, BL21(DE3) carrying cenA gene could decompose CMC-Na while negative control couldn't, which proved that cenA could work successfully.

References