Difference between revisions of "Part:BBa K1149007"

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.

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.

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]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1149007 short</partinfo>
-Encodes a peptidase from proteinase K family, breaks down polylactic acid.
+Encodes a peptidase from proteinase K family, breaks down polylactic acid (no PelB secretion tag).
-The [https://parts.igem.org/Part:BBa_K1149008 BBa_K1149008] construct is the same enzyme but wit the pelB secretion tag.
+The [https://parts.igem.org/Part:BBa_K1149008 BBa_K1149008] construct is the same enzyme but with the pelB secretion tag.
-<p>.</p>
+<h2>Characterisation</h2>
-reference: 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.
+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.
+https://static.igem.org/mediawiki/2013/thumb/3/32/PK.png/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.
+https://static.igem.org/mediawiki/2013/thumb/6/6c/PK_fluor.png/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.
+<b>Conclusion: There is no growth inhibition caused by induction, but fluorescence from induction is elevated in Proteinase K (intracellular). </b>
+<h2>References: </h2>
+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