Difference between revisions of "Part:BBa K118023"
(→Congo Red Assay) |
|||
| Line 14: | Line 14: | ||
===Molecular weight=== | ===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 | + | ===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 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. | ||
Revision as of 16:54, 13 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 34
Illegal NotI site found at 1178 - 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 369
Illegal NgoMIV site found at 1294 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 273
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 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.
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.
3,5-dinitrosalicylic acid (DNS) method
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).
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.