Difference between revisions of "Part:BBa K3606809"
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McbD works as synthase responsible for the maturation of MccB17.
 
McbD works as synthase responsible for the maturation of MccB17.
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<h2>Design:</h2>
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Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we decided to check whether the McbA/B/C/D/E/F/G genes can be expressed and translated separately. We successfully cloned McbG into the pGEX plasmid and transcribed it into BL21. After 5 hours of induction of McbG expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbG product was produced.
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<h2>Results:</h2>
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https://2020.igem.org/wiki/images/2/22/T--Fudan--img_McbA-E-G-BCD.jpeg
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Figure 1. SDS-PAGE result. lane 1: mcbA  lane 2: mcbA+IPTG lane 3: mcbE  lane 4: mcbE+IPTG  lane 5: mcbG  lane 6: mcbG+IPTG  lane 7: mcbBCD  lane 8: mcbBCD+IPTG lane 9: pGEX+ IPTG
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Comparing the 5th and 6th lanes, after IPTG induction, there is a clear band, which is the product of McbG expression.
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Revision as of 01:34, 28 October 2020


mcbD

McbD works as synthase responsible for the maturation of MccB17.

Design:

Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we decided to check whether the McbA/B/C/D/E/F/G genes can be expressed and translated separately. We successfully cloned McbG into the pGEX plasmid and transcribed it into BL21. After 5 hours of induction of McbG expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbG product was produced.

Results:

T--Fudan--img_McbA-E-G-BCD.jpeg

Figure 1. SDS-PAGE result. lane 1: mcbA lane 2: mcbA+IPTG lane 3: mcbE lane 4: mcbE+IPTG lane 5: mcbG lane 6: mcbG+IPTG lane 7: mcbBCD lane 8: mcbBCD+IPTG lane 9: pGEX+ IPTG

Comparing the 5th and 6th lanes, after IPTG induction, there is a clear band, which is the product of McbG expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 374
    Illegal PstI site found at 407
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 374
    Illegal PstI site found at 407
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 281
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 374
    Illegal PstI site found at 407
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 374
    Illegal PstI site found at 407
    Illegal NgoMIV site found at 36
    Illegal AgeI site found at 208
  • 1000
    COMPATIBLE WITH RFC[1000]