Part:BBa_K2553002
pLuxlacO_CDA1
chitin deacetylase 1 from Penaeus Monodon. This enzyme can widely be found in nature. This part has the down stream of Qorum sensing system as the subpart, corresponding to BBa K2553007.
Chitin deacetylase (CDA) could hydrolyze the acetamino group on chitin, directly yielding chitin. As reported, CDA has been found in bacteria, moulds and insects, but the examined ones are all inactive toward crystallized chitin, whereas the only industrial-available source of chitin is the especially high-crystallized chitin from shrimp and crab shell. This is the key problem of the application of enzymolysis method to industry. Separation, identification and production of crystal-chitin-active-enzymes is crucial to solving this problem, hence accelerating the industrialization of environmental-friendly chitosan production considerably, and give instruction to research on arthropodic ecdysis and aquacultural production.
Using the QS system to control transcription can improve both the efficiency and stability of the target protein's transcription. The autoinducing mechanism saves the effort of using artificial inducers such as IPTG, and reduced steps also mean fewer things can go wrong. Also, this system is self-regulating, which means that the colony will automatically adjust to ensure stable production without the need for interference from researchers.
For those aiming to work with QS system, this construction offers an easy way to evaluate the performance of the system.
Characterization by ultraviolet
1.Amplification DNA amplification it with BBa K2553007.
2. Double Enzyme Digestion (1) Conduct Double Enzyme Digestion on the fragments and original PET-28a, and operate electrophoresis. The target bands are collected and recovered. (2) The bands are mixed with the pEt 28-a plasmid as a ratio of 9:1, and the instruction were carried out using T4 ligase kit at 16 degrees for 1-4 hours. (3) Then, the ligated product can be directly transformed into a BL-21 expression strain.
3. Transfection The plasmid was diluted 1000-fold and 10000-fold, and the competent bacteria BL-21 was added for 30 minutes in an ice bath. Then we heated the bacteria at 42 degrees for one minute, and ice-bathed for five more minutes. After that, we added 400 ml of LB without Kana antibiotic and shook the bacterial fluid. Finally, the bacteria were sprayed on a petri dish with kana antibiotics.
4.Test Enzyme Activity We add 1 mL 200 m g / L of substrate to a clean test tube, 3mL 0.05 mol / L phosphate buffer , 3 min 50 ° C water bath temperature protection Min, and add 1 mL enzyme solution, mix and heat in 50 degrees water 15 min. After that, we used boiling water to terminate the enzymatic reaction, and add water to a volume of 10 mL. Evenly, the solution was centrifuged at 3000r /min for 10 min. Measure the OD value of the upper clear.
Definition of enzyme unit: The amount of enzyme required to produce 1 gram of p-nitroaniline per hour under the above reaction conditions is defined as 1 enzyme activity ( U /m L ).
Length Wave CDA1-1 CDA1-2 pET28a-1 pET28a-2 400 0.03 0.07 -0.001 0.004
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1315
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1315
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1315
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1315
Illegal NgoMIV site found at 244 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 544
Illegal BsaI.rc site found at 1435
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