Difference between revisions of "Part:BBa K2933129"

 
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<partinfo>BBa_K2933129 parameters</partinfo>
 
<partinfo>BBa_K2933129 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein MUS-2. It encodes a protein which is MUS-2 fused with His-Sumo tag. The fusion protein is about 40.3kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of MUS-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.
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===Molecular cloning===
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We used the vector pGEX-6p-1 to construct our expression plasmid.
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<p style="text-align: center;">
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  [[File:TJUSLS China--MUS-2-PCR.png|500px]]<br>
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'''Figure 1.'''  Left: The PCR result of MUS-2. Right: The verification results by enzyme digestion.<br>
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</p>
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After verification, it was determined that the construction is successful.
 +
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===References===
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[1]Al-Bayssari C, Gupta SK, Dabboussi F, Hamze M, Rolain JM. MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus. New Microbes New Infect. 2015 Jun 27;7:67-71.
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[2] Mammeri H, Bellais S, Nordmann P. Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes. Antimicrob Agents Chemother. 2002 Nov;46(11):3561-7.

Revision as of 13:37, 24 September 2019


His+Linker a+Sumo+Linker b+MUS-2

This part encodes the fusion protein of His tag, sumo tag and MUS-2 to promote the expression and purification of target protein(MUS-2).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein MUS-2. It encodes a protein which is MUS-2 fused with His-Sumo tag. The fusion protein is about 40.3kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of MUS-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

Molecular cloning

We used the vector pGEX-6p-1 to construct our expression plasmid.

TJUSLS China--MUS-2-PCR.png
Figure 1. Left: The PCR result of MUS-2. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful.

References

[1]Al-Bayssari C, Gupta SK, Dabboussi F, Hamze M, Rolain JM. MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus. New Microbes New Infect. 2015 Jun 27;7:67-71.

[2] Mammeri H, Bellais S, Nordmann P. Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes. Antimicrob Agents Chemother. 2002 Nov;46(11):3561-7.