Difference between revisions of "Part:BBa K2013003"
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https://static.igem.org/mediawiki/2016/9/97/T--UESTC-China--BBa_K2013003-Double_digestion.png
 
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==Expression of enzymes==
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===SDS-PAGE===
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The extracellular expression level of PETase was tested from the supernatant fractions collected from E. coli culture after 4h incubation by SDS-PAFE analysis. Surprisingly, we found that our targeted band represents the dominant band in the agarose gel. The results might indicate that the extracellular expression level reached at maximum at 4h. With prolonged incubation time, host cells produced more extracellular proteins and might interfere with the function of PETase.
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https://static.igem.org/mediawiki/2016/8/8a/T--UESTC-China--SDS123567puc.jpeg
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M: protein marker;Line1:piGEM2016-001;Line2:piGEM2016-002;
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Line3:piGEM2016-003;Line4:piGEM2016-005;Line5:piGEM2016-006;
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Line6:piGEM2016-007; Line7:pUC57; Line8: E. coli BL21 (DE3)
  
  

Revision as of 05:25, 21 October 2016


RBS and MHETase

This part contains the coding sequence of MHETase which can hydrolyzes MHET to TPA and EG. MHETase is second enzymes on the downstream of PETase in the bacterium Ideonella sakaiensis 201-F6 that Japanese scientists found.MHET is degraded into two kinds of natural environment harmless substances: terephthalic acid and ethylene glycol. 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 579
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 264
    Illegal NgoMIV site found at 378
    Illegal NgoMIV site found at 834
    Illegal AgeI site found at 331
    Illegal AgeI site found at 448
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental Validation

This part is validated through four ways: amplification, PCR, Enzyme cutting, SDS-PAGE and Sequence.

Amplification

Enzyme:Q5

Primer-F:5′- GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAAAAGGGTACTAGATG-3′

Primer-R:5′- CGCTACTAGTATTATTACGGCGGAGCCGCGCAC-3′

Results

T--UESTC-China--BBa_K2013003-Amplification.png


PCR

Enzyme:Taq

Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′

Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′

Results

T--UESTC-China--BBa_K2013003-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 HindIII restriction endonuclease. The second cutting procedure was performed with PstI and BaHI 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_K2013003-Double_digestion.png

Expression of enzymes

SDS-PAGE

The extracellular expression level of PETase was tested from the supernatant fractions collected from E. coli culture after 4h incubation by SDS-PAFE analysis. Surprisingly, we found that our targeted band represents the dominant band in the agarose gel. The results might indicate that the extracellular expression level reached at maximum at 4h. With prolonged incubation time, host cells produced more extracellular proteins and might interfere with the function of PETase.

T--UESTC-China--SDS123567puc.jpeg

M: protein marker;Line1:piGEM2016-001;Line2:piGEM2016-002;

Line3:piGEM2016-003;Line4:piGEM2016-005;Line5:piGEM2016-006;

Line6:piGEM2016-007; Line7:pUC57; Line8: E. coli BL21 (DE3)