Part:BBa_K2013015
protocatechuate decarboxylase
This part contains the sequence of coding protocatechuate decarboxylase which catalyzes protocatechuate into catechol.In addition，we did codon optimization before synthesizing it to encode the target product successfully in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 208
Illegal NgoMIV site found at 511
Illegal NgoMIV site found at 787
Illegal AgeI site found at 524
Illegal AgeI site found at 701 - 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:KOD
Primer-F:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACATGAGGGTACTAGATG-3′
Primer-R:5′- CGCTACTAGTATTATTAGCTTTGCAGGTAAACCAC-3′
Results
PCR
Enzyme:Taq
Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′
Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′
Results
Double digestion
After the assembly ,the plasmid was transferred into the Competent E. coli top10. After culturing overnight in LB,we minipreped the plasmid for double digestion .The first cutting procedure was performed with EcoRI and NcoI restriction endonuclease. The second cutting procedure was performed with PstI and NcoI restriction endonuclease.The plasmid was cutted in a 25μL system at 37 ℃ for 1 hours. The Electrophoresis was performed on a 1％ Agarose glu
Results
| None |