Translational_Unit

Part:BBa_K2013005

Designed by: Binglin Liu   Group: iGEM16_UESTC-China   (2016-10-12)


RBS and terephthalic acid(TPA) transporter

There are three sequences co-encoding TPA transporter.The sequence(ISF6_0077) in this part is the second. Because of it,TPA will be transported from extracellular to intracellular.In addition, we did codon optimization before synthesizing it to encode the target product successfully in E. coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Experimental Validation

This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.

Amplification

Enzyme:Q5

Primer-F:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAAAAGGGTACTAGATG-3′

Primer-R:5′- CGCTACTAGTATTATTAATCTTCTTGAAACGCGTCCT-3′

Results

T--UESTC-China--BBa_K2013005-Amplification.png

PCR

Enzyme:Taq

Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′

Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′

Results

T--UESTC-China--BBa_K2013005-PCR.png

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 SpeI restriction endonuclease. The second cutting procedure was performed with NcoI and PstI 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

T--UESTC-China--BBa_K2013005-Double_digestion.png

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