Difference between revisions of "Part:BBa K1582021"
 
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<partinfo>BBa_K1582021 parameters</partinfo>
 
<partinfo>BBa_K1582021 parameters</partinfo>
 
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===Usage===
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This fusion protein is designed to degrade PET into terephthalic acid and ethylene glycol more powerfully. Compared to single FsC, the rate of PET emzymolysis is supposed to be improved by LC-Janus fusion.
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===Biology===
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FsC is a kind of cutinase, the details of which can be seen at BBa_K808025. Class II hydrophobins sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. Their assembly shows thread-like structure. They could be expressed in prokaryotic cells like E.coli. We did some mutations to it, and we call it sJanus-m.<br>
 +
According to some research, when Janus is added to the reaction of cutinase and plastics, the reaction rate is supposed to have an obvious improvement.<br>
 +
Based on the above background, we design to use the method of fusion protein to combine cutinase and Janus compactly, through the establishment of fusion protein, we can found if we can further promote the stimulation rate of the plastics degradation. Meanwhile, from the data about fusion protein, we can try to uncover the functional mechanism of the stimulation to the plastics degradation which is unknown until now. <br>
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===Protein Expression===
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Inoculate of 1L liquid LB-media with C+_FsC-sJanus_pET-28a, C+_FsC-sJanus-m_pET-28a for 4h. Add 1M IPTG 500μL and induce at 16°C for 15h. Centrifuge of 1L LB medium at 4000rpm for 20min and discard supernatant. Crush bacteria with High Pressure Homogenizer and centrifuge at 18000rpm for 30min. Then purify protein with nickel column and we get the FsC-sJanus and FsC-sJanus-m.
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<p style="text-align: center;">
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    https://static.igem.org/mediawiki/2015/d/d7/Tianjin_result07.png<br>
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'''Figure 1.''' The result of protein C+_FsC-sJanus-m expression. M is Protein marker. a is sample of FsC-sJanus-m in C+ which is non-induced. b is sample of FsC-sJanus-m in C+ which is induced. c is sample of sediment after breaking bacteria and centrifugation. d if sample of liquid after filtration by Ni column. e is sample of media after filtration by Ni column. f is sample of liquid after removing impurity with 50mM MCAC. g is sample of media after removing impurity with 50mM MCAC. h is sample of liquid after washing with 200mM MCAC. i is sample of media after washing with 200mM MCAC. j is sample of target protein after washing with 200mM MCAC.
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</p>

Latest revision as of 17:12, 21 September 2015

FsC+sJanus-m Fusion Protein


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 379
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 525
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 907
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 301
    Illegal SapI site found at 916


Usage

This fusion protein is designed to degrade PET into terephthalic acid and ethylene glycol more powerfully. Compared to single FsC, the rate of PET emzymolysis is supposed to be improved by LC-Janus fusion.

Biology

FsC is a kind of cutinase, the details of which can be seen at BBa_K808025. Class II hydrophobins sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. Their assembly shows thread-like structure. They could be expressed in prokaryotic cells like E.coli. We did some mutations to it, and we call it sJanus-m.
According to some research, when Janus is added to the reaction of cutinase and plastics, the reaction rate is supposed to have an obvious improvement.
Based on the above background, we design to use the method of fusion protein to combine cutinase and Janus compactly, through the establishment of fusion protein, we can found if we can further promote the stimulation rate of the plastics degradation. Meanwhile, from the data about fusion protein, we can try to uncover the functional mechanism of the stimulation to the plastics degradation which is unknown until now.

Protein Expression

Inoculate of 1L liquid LB-media with C+_FsC-sJanus_pET-28a, C+_FsC-sJanus-m_pET-28a for 4h. Add 1M IPTG 500μL and induce at 16°C for 15h. Centrifuge of 1L LB medium at 4000rpm for 20min and discard supernatant. Crush bacteria with High Pressure Homogenizer and centrifuge at 18000rpm for 30min. Then purify protein with nickel column and we get the FsC-sJanus and FsC-sJanus-m.

Tianjin_result07.png
Figure 1. The result of protein C+_FsC-sJanus-m expression. M is Protein marker. a is sample of FsC-sJanus-m in C+ which is non-induced. b is sample of FsC-sJanus-m in C+ which is induced. c is sample of sediment after breaking bacteria and centrifugation. d if sample of liquid after filtration by Ni column. e is sample of media after filtration by Ni column. f is sample of liquid after removing impurity with 50mM MCAC. g is sample of media after removing impurity with 50mM MCAC. h is sample of liquid after washing with 200mM MCAC. i is sample of media after washing with 200mM MCAC. j is sample of target protein after washing with 200mM MCAC.