Difference between revisions of "Part:BBa K5241006"

 
 
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<partinfo>BBa_K5241006 short</partinfo>
 
<partinfo>BBa_K5241006 short</partinfo>
  
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Encodes a fusion protein consisting of the Pkrhdd enzyme from Pseudomonas koreensis, an N-terminal T7 tag for solubility, and a C-terminal 6xHis tag for purification. The fusion protein is under the control of the pBAD promoter, which allows for arabinose-inducible expression, and confers ampicillin resistance (AmpR).
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<span style="font-weight: bold;">Short Description:</span>Encodes a fusion protein consisting of the Pkrhdd enzyme from Pseudomonas koreensis, an N-terminal T7 tag for solubility, and a C-terminal 6xHis tag for purification. The fusion protein is under the control of the pBAD promoter, which allows for arabinose-inducible expression, and confers ampicillin resistance (AmpR).
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<figure><img style="width: 65%; padding:28px;"src="https://static.igem.wiki/teams/5241/wiki/parts-static/6/bba-k5241006-img01.png">
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<figcaption>araBAD promoter 6xHis T7 tag pBAD-6xH-pelBPkrhdd-6xH(containing pelB signal and 6xHis）rrnB T1 terminator</figcaption>
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<span style="font-weight: bold;">Source:</span>Pseudomonas koreensis,
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<span style="font-size: 150%;font-weight: bold;">Description：</span>
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Using the designed primers, perform PCR reactions with templates containing the target genes (pelB, Pkhdd) from Pseudomonas koreensis to amplify the required gene fragments. Select appropriate restriction enzymes to digest the pBAD plasmid, creating sticky ends that match the target gene fragments. Purify the target gene fragments and ligate them with the digested pBAD plasmid using DNA ligase. The target gene fragments combine with the plasmid backbone through complementary sticky ends to form the complete plasmid pBAD-pelBPkhdd. Transform the ligation product into the suitable host cells - Escherichia coli TOP10.
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<span style="font-size: 150%; font-weight: bold;">Result:</span>
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The PBAD vector contains the arabinose operon, and the absence of melanin production after induction with L-arabinose indicates that the construction of the PBAD-6XH-PelBPkrhdd plasmid has failed.
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<figure><img style="width: 65%; padding:28px;"src="https://static.igem.wiki/teams/5241/wiki/parts-static/6/bba-k5241006-img02.png"></figure>
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</html>
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<span style="font-size: 150%;font-weight: bold;">Reference documentation</span>
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[1] Clark, M. A., Hammond, F. R., Papaioannou, A., Hawkins, N. J., & Ward, R. L. (1997). Design of a novel switchable antibody display system in Pichia pastoris. Immunotechnology, 3(4), 215-223. https://doi.org/10.1016/s1380-2933(97)00016-x
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[2] Schuller, Artur, et al. "Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems." Microb. Cell Fact., vol. 19, no. 1, 5 Mar. 2020, p. 6209-6224, https://doi.org/10.1186/s12934-020-01311-6
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 05:21, 30 September 2024


pBAD-6xH-pelBPkrhdd-6xH

Short Description:Encodes a fusion protein consisting of the Pkrhdd enzyme from Pseudomonas koreensis, an N-terminal T7 tag for solubility, and a C-terminal 6xHis tag for purification. The fusion protein is under the control of the pBAD promoter, which allows for arabinose-inducible expression, and confers ampicillin resistance (AmpR).

araBAD promoter 6xHis T7 tag pBAD-6xH-pelBPkrhdd-6xH(containing pelB signal and 6xHis）rrnB T1 terminator

Source:Pseudomonas koreensis,

Description：

Using the designed primers, perform PCR reactions with templates containing the target genes (pelB, Pkhdd) from Pseudomonas koreensis to amplify the required gene fragments. Select appropriate restriction enzymes to digest the pBAD plasmid, creating sticky ends that match the target gene fragments. Purify the target gene fragments and ligate them with the digested pBAD plasmid using DNA ligase. The target gene fragments combine with the plasmid backbone through complementary sticky ends to form the complete plasmid pBAD-pelBPkhdd. Transform the ligation product into the suitable host cells - Escherichia coli TOP10.

Result:

The PBAD vector contains the arabinose operon, and the absence of melanin production after induction with L-arabinose indicates that the construction of the PBAD-6XH-PelBPkrhdd plasmid has failed.

Reference documentation

[1] Clark, M. A., Hammond, F. R., Papaioannou, A., Hawkins, N. J., & Ward, R. L. (1997). Design of a novel switchable antibody display system in Pichia pastoris. Immunotechnology, 3(4), 215-223. https://doi.org/10.1016/s1380-2933(97)00016-x

[2] Schuller, Artur, et al. "Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems." Microb. Cell Fact., vol. 19, no. 1, 5 Mar. 2020, p. 6209-6224, https://doi.org/10.1186/s12934-020-01311-6


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal PstI site found at 925
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal PstI site found at 925
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal XhoI site found at 1075
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal PstI site found at 925
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal PstI site found at 925
    Illegal NgoMIV site found at 342
    Illegal NgoMIV site found at 1140
    Illegal AgeI site found at 319
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 316