Part:BBa_K2013009
TPA 1,2-dioxygenase
This part contains one of sequences(ISF6_0228) that relates to encode TPA 1,2-dioxygenase.This is the second. TPA is incorporated through the TPA transporter (TPATP) and catabolized by TPA 1,2-dioxygenase (TPADO).The result is that TPA is catabolized into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate.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 AgeI site found at 299
Illegal AgeI site found at 347 - 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′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAGGAGAGTACTAGATG-3′
Primer-R:5′- CGCTACTAGTATTATTACAGCGGCAGCGCCA-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 EcoRV 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 |