Part:BBa_K2013013
PCA 3.4-dioxygenase
This part contains one of sequences(ISF6_0627) that relates to encode Protocatechuate(PCA)3.4-dioxygenase.when PCA3.4-dioxygenase works, the resultant Protocatechuate will be ring-cleaved.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 79
Illegal NgoMIV site found at 91
Illegal NgoMIV site found at 766
Illegal AgeI site found at 167 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 632
Experimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:KOD
Primer-F:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAGGAGGGTACTAGATG-3′
Primer-R:5′- CGCTACTAGTATTATTAACGCGCATCACCCGCCG-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 EcoRV 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 |