Part:BBa_K5193002
exoglucanase and endoglucanase
It is a combination of two cellulases: exoglucanase and endoglucanase. By inserting the two genes into the Multiple Cloning Site (MCS) blocks (with separate, two P7 promoters), the engineered bacteria can therefore produce two types of enzyme in huge amounts at the same time. We used the two enzymes to break down cellulose in plant cell walls [1] , thereby releasing a greater amount of essential oil.
The activity of cellulase was measured by DNS (3,5-dinitrosalicylic acid) method through the amount of reducing sugars liberated during hydrolysis.[2]
After adding CMC (Carboxymethyl cellulose solution) into our cell culture, we first incubated the solution for 2 hours at room temperature, 50C and 90C in order to let the reaction take place. But we found out that the most significant impact on the result is when the incubation takes place at 50C.(See fig 1.) Therefore we chose to incubate the solution for 2 hours in 50C. After adding DNS reagent to the solution, we incubated the solution again for another 10 minutes in 50C to stop the reaction. We then add our solution into a 96-well transparent plate for OD measurement at 540 nm. Results are shown below.
References: 1. Islam F, Roy N. Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. Islam and Roy BMC Res Notes (2018) 11:445 https://doi.org/10.1186/s13104-018-3558-4 2. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31: 426-428. 10.1021/ac60147a030
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 524
Illegal NotI site found at 1501
Illegal NotI site found at 2645 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2558
Illegal BamHI site found at 1694
Illegal XhoI site found at 2056
Illegal XhoI site found at 2305 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 157
Illegal NgoMIV site found at 530
Illegal NgoMIV site found at 1032
Illegal NgoMIV site found at 1836
Illegal NgoMIV site found at 2761 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1740
Illegal BsaI.rc site found at 577
Illegal SapI.rc site found at 660
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