Difference between revisions of "Part:BBa K1149007"

 
 
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__NOTOC__
 
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<partinfo>BBa_K1149007 short</partinfo>
 
<partinfo>BBa_K1149007 short</partinfo>
  
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Encodes a peptidase from proteinase K family, breaks down polylactic acid.
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Encodes a peptidase from proteinase K family, breaks down polylactic acid (no PelB secretion tag).
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The BBa_K1149008 construct is the same enzyme but wit the pelB secretion tag.
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The [https://parts.igem.org/Part:BBa_K1149008 BBa_K1149008] construct is the same enzyme but with the pelB secretion tag.
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<h2>Characterisation</h2>
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Our Proteinase K (no PelB secretion tag) construct contains sfGFP within an operon and therefore fluorescence can be utilised to determine if expression is being induced by addition of Arabinose.
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https://static.igem.org/mediawiki/2013/thumb/3/32/PK.png/450px-PK.png
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'''Proteinase K growth assay.''' MG1655 ''E. coli'' display strong growth with the induction of BBa_K1149007, though it is not significant as a two-tailed t-test gave p = 0.1471. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.
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https://static.igem.org/mediawiki/2013/thumb/6/6c/PK_fluor.png/450px-PK_fluor.png
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'''Proteinase K induction assay.''' When induced, there is a very clear increase in fluorescence for BBa_K1149007, however t-test analysis gives p = 0.0595 > 0.05 for this, thus the fluorescence does not significantly differ from uninduced. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.
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<b>Conclusion: There is no growth inhibition caused by induction, but fluorescence from induction is elevated in Proteinase K (intracellular). </b>
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<h2>References: </h2>
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Larsen AN, Moe E, Helland R, Gjellesvik DR, Willassen NP.Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp. FEBS J. 2006 Jan;273(1):47-60.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 23:43, 2 October 2013

Proteinase K

Encodes a peptidase from proteinase K family, breaks down polylactic acid (no PelB secretion tag). The BBa_K1149008 construct is the same enzyme but with the pelB secretion tag.

Characterisation

Our Proteinase K (no PelB secretion tag) construct contains sfGFP within an operon and therefore fluorescence can be utilised to determine if expression is being induced by addition of Arabinose.

450px-PK.png

Proteinase K growth assay. MG1655 E. coli display strong growth with the induction of BBa_K1149007, though it is not significant as a two-tailed t-test gave p = 0.1471. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

450px-PK_fluor.png

Proteinase K induction assay. When induced, there is a very clear increase in fluorescence for BBa_K1149007, however t-test analysis gives p = 0.0595 > 0.05 for this, thus the fluorescence does not significantly differ from uninduced. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: There is no growth inhibition caused by induction, but fluorescence from induction is elevated in Proteinase K (intracellular).

References:

Larsen AN, Moe E, Helland R, Gjellesvik DR, Willassen NP.Characterization of a recombinantly expressed proteinase K-like enzyme from a psychrotrophic Serratia sp. FEBS J. 2006 Jan;273(1):47-60.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
    Illegal NheI site found at 943
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 290
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1465
  • 1000
    COMPATIBLE WITH RFC[1000]