http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&feed=atom&action=historyTeam:Heidelberg/pages/Linker Screening - Revision history2024-03-29T10:13:47ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=403743&oldid=prevMaexlich at 16:56, 8 December 20142014-12-08T16:56:53Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Cloning of the expression constructs==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Cloning of the expression constructs==</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by <del class="diffchange diffchange-inline">CPEC </del>[<del class="diffchange diffchange-inline">REF TO MAT</del>/<del class="diffchange diffchange-inline">MET </del>CPEC] from PCR products of [http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by [<ins class="diffchange diffchange-inline">https:</ins>/<ins class="diffchange diffchange-inline">/2014.igem.org/Team:Heidelberg/Notebook/Methods#CPEC </ins>CPEC] from PCR products of [http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to create the construct to express linear lambda lysozyme with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between the lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction of the vector PCR product was designed and ordered as DNA-oligos. The insert was annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [<del class="diffchange diffchange-inline">REF TO MAT</del>/<del class="diffchange diffchange-inline">MET </del>Golden Gate assembly].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to create the construct to express linear lambda lysozyme with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between the lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction of the vector PCR product was designed and ordered as DNA-oligos. The insert was annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [<ins class="diffchange diffchange-inline">https://2014.igem.org/Team:Heidelberg/Notebook</ins>/<ins class="diffchange diffchange-inline">Methods#Golden_Gate_Assembly </ins>Golden Gate assembly].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The constructs to express circular lambda lysozyme with a rigid linker [http://parts.igem.org/Part:BBa_K1362013 (BBa_K1362013)], a His6 linker [http://parts.igem.org/Part:BBa_K1362012 (BBa_K1362012)] and all other linkers in shown in table 1 are based on the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(<del class="diffchange diffchange-inline">i</del>) circularization construct]. These cloning steps were carried out according to our new RFC[<del class="diffchange diffchange-inline">i</del>].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The constructs to express circular lambda lysozyme with a rigid linker [http://parts.igem.org/Part:BBa_K1362013 (BBa_K1362013)], a His6 linker [http://parts.igem.org/Part:BBa_K1362012 (BBa_K1362012)] and all other linkers in shown in table 1 are based on the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(<ins class="diffchange diffchange-inline">105</ins>) circularization construct]. These cloning steps were carried out according to our new RFC[<ins class="diffchange diffchange-inline">105</ins>].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For [http://parts.igem.org/Part:BBa_K1362013 BBa_K1362013] and [http://parts.igem.org/Part:BBa_K1362012 BBa_K1362012], the coding sequence of lambda lysozyme without start and stop codon was aquired from genomic lambda DNA by PCR. Overhangs containing a BsaI site, the RFC[<del class="diffchange diffchange-inline">i</del>] E standard overhang, the coding sequences of the extein CWE (forward) and the corresponding linker sequence, RGK, the RFC[<del class="diffchange diffchange-inline">i</del>] B standard overhang, a BsaI site (reverse) were added by primers. The PCR products were purified and inserted each into the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(<del class="diffchange diffchange-inline">i</del>) circularization construct] by Golden Gate assembly.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For [http://parts.igem.org/Part:BBa_K1362013 BBa_K1362013] and [http://parts.igem.org/Part:BBa_K1362012 BBa_K1362012], the coding sequence of lambda lysozyme without start and stop codon was aquired from genomic lambda DNA by PCR. Overhangs containing a BsaI site, the RFC[<ins class="diffchange diffchange-inline">105</ins>] E standard overhang, the coding sequences of the extein CWE (forward) and the corresponding linker sequence, RGK, the RFC[<ins class="diffchange diffchange-inline">105</ins>] B standard overhang, a BsaI site (reverse) were added by primers. The PCR products were purified and inserted each into the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(<ins class="diffchange diffchange-inline">105</ins>) circularization construct] by Golden Gate assembly.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] and two inserts with subsequent [<del class="diffchange diffchange-inline">REF TO MAT</del>/<del class="diffchange diffchange-inline">MET </del>religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[<del class="diffchange diffchange-inline">i</del>] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[<del class="diffchange diffchange-inline">i</del>] B standard overhang and a BsaI site (reverse) were added by primers.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] and two inserts with subsequent [<ins class="diffchange diffchange-inline">https:</ins>/<ins class="diffchange diffchange-inline">/2014.igem.org/Team:Heidelberg/Notebook/Methods#Religation_after_Golden_gate_Assembly </ins>religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[<ins class="diffchange diffchange-inline">105</ins>] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[<ins class="diffchange diffchange-inline">105</ins>] B standard overhang and a BsaI site (reverse) were added by primers.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly. </div></td></tr>
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</table>Maexlichhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=389143&oldid=prevJan glx: /* Results */2014-10-18T02:29:46Z<p><span class="autocomment">Results</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>caption=Figure 7, Activity after heatshock|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>caption=Figure 7, Activity after heatshock|</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>align=right |</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>align=right |</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The <del class="diffchange diffchange-inline">errors are estimations </del>from the <del class="diffchange diffchange-inline">standard deviations of </del>the different replicates. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The <ins class="diffchange diffchange-inline">single points come </ins>from <ins class="diffchange diffchange-inline">different technical and biological replicates. For clarification a simple spline was fitted to </ins>the <ins class="diffchange diffchange-inline">data. The fits produced 95% confidence intervals, that would exceed the scale. But as on can see that mostly </ins>the different replicates <ins class="diffchange diffchange-inline">don't scatter too much, these errors are not too relevant</ins>. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>file=resultsofscreening_new.png}} </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>file=resultsofscreening_new.png}} </div></td></tr>
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</table>Jan glxhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=388575&oldid=prevJan glx: /* Results */2014-10-18T02:24:56Z<p><span class="autocomment">Results</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>align=right |</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>align=right |</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The errors are estimations from the standard deviations of the different replicates. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The errors are estimations from the standard deviations of the different replicates. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>file=<del class="diffchange diffchange-inline">resultsaveragesofscreening</del>.png}} </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>file=<ins class="diffchange diffchange-inline">resultsofscreening_new</ins>.png}} </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further clarification we have calculated the average activity between 45 and 50°C, because there the biggest change in activity could be observed for the different constructs. Furthermore the values of the activities vary a lot, what can especially be seen at circsho1 linker. This averaging was made for clarification and for obtaining a final ranking of the linkers to feed back to the linker software (see table 2)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For further clarification we have calculated the average activity between 45 and 50°C, because there the biggest change in activity could be observed for the different constructs. Furthermore the values of the activities vary a lot, what can especially be seen at circsho1 linker. This averaging was made for clarification and for obtaining a final ranking of the linkers to feed back to the linker software (see table 2)</div></td></tr>
</table>Jan glxhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=372467&oldid=prevMaxW at 00:04, 18 October 20142014-10-18T00:04:38Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by CPEC [REF TO MAT/MET CPEC] from PCR products of [http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by CPEC [REF TO MAT/MET CPEC] from PCR products of [http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to create the construct to express linear lambda lysozyme with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between the lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction of the vector PCR product was designed and ordered as DNA-oligos. The insert was annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [REF TO MAT/MET Golden Gate assembly].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to create the construct to express linear lambda lysozyme with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between the lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction of the vector PCR product was designed and ordered as DNA-oligos. The insert was annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [REF TO MAT/MET Golden Gate assembly].</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] and two inserts with subsequent [REF TO MAT/MET religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[i] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[i] B standard overhang and a BsaI site (reverse) were added by primers.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] and two inserts with subsequent [REF TO MAT/MET religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[i] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[i] B standard overhang and a BsaI site (reverse) were added by primers.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly. </div></td></tr>
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</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=372319&oldid=prevMaxW at 00:03, 18 October 20142014-10-18T00:03:28Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Experimental procedure=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Experimental procedure=</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==Cloning of <del class="diffchange diffchange-inline">lysozyme fused to linkers</del>==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==Cloning of <ins class="diffchange diffchange-inline">the expression constructs</ins>==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">To get the circular form </del>of lambda lysozyme <del class="diffchange diffchange-inline">we used </del>the <del class="diffchange diffchange-inline">autocatalytic function </del>of the <del class="diffchange diffchange-inline">intein NpuDnaE, an often used </del>and <del class="diffchange diffchange-inline">well</del>-<del class="diffchange diffchange-inline">described split-intein</del>. <del class="diffchange diffchange-inline">Lambda lysozyme </del>was <del class="diffchange diffchange-inline">cloned </del>in our new standard of <del class="diffchange diffchange-inline">circularization</del>, <del class="diffchange diffchange-inline">described </del>[<del class="diffchange diffchange-inline">https</del>://<del class="diffchange diffchange-inline">2014</del>.igem.org/<del class="diffchange diffchange-inline">Team</del>:<del class="diffchange diffchange-inline">Heidelberg</del>/<del class="diffchange diffchange-inline">Toolbox</del>/<del class="diffchange diffchange-inline">Circularization here</del>].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The construct to express linear lambda lysozyme [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] was assembled by CPEC [REF TO MAT/MET CPEC] from PCR products </ins>of <ins class="diffchange diffchange-inline">[http://parts.igem.org/Part:BBa_K1362093 pSBX1K30] and phage lambda genome. The RBS [http://parts.igem.org/Part:BBa_K1362090 BBa_K1362090] was added by primer overhangs.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">In order to create the construct to express linear </ins>lambda lysozyme <ins class="diffchange diffchange-inline">with His6-tag and exteins, primers to linearize [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] between </ins>the <ins class="diffchange diffchange-inline">lambda lysozyme coding sequence and stop codon and add BsaI sites at the ends were designed. An insert coding for the sequence GSHHHHHHSGRGKCWE (His6-tag + exteins) with overhangs corresponding to the overhangs caused by BsaI restriction </ins>of the <ins class="diffchange diffchange-inline">vector PCR product was designed </ins>and <ins class="diffchange diffchange-inline">ordered as DNA</ins>-<ins class="diffchange diffchange-inline">oligos</ins>. <ins class="diffchange diffchange-inline">The insert </ins>was <ins class="diffchange diffchange-inline">annealed and inserted into the purified PCR product of [http://parts.igem.org/Part:BBa_K1362011 BBa_K1362011] by [REF TO MAT/MET Golden Gate assembly].</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The constructs to express circular lambda lysozyme with a rigid linker [http://parts.igem.org/Part:BBa_K1362013 (BBa_K1362013)], a His6 linker [http://parts.igem.org/Part:BBa_K1362012 (BBa_K1362012)] and all other linkers </ins>in <ins class="diffchange diffchange-inline">shown in table 1 are based on the [http://parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct]. These cloning steps were carried out according to </ins>our new <ins class="diffchange diffchange-inline">RFC[i].</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For [http://parts.igem.org/Part:BBa_K1362013 BBa_K1362013] and [http://parts.igem.org/Part:BBa_K1362012 BBa_K1362012], the coding sequence of lambda lysozyme without start and stop codon was aquired from genomic lambda DNA by PCR. Overhangs containing a BsaI site, the RFC[i] E </ins>standard <ins class="diffchange diffchange-inline">overhang, the coding sequences </ins>of <ins class="diffchange diffchange-inline">the extein CWE (forward) and the corresponding linker sequence</ins>, <ins class="diffchange diffchange-inline">RGK, the RFC[i] B standard overhang, a BsaI site (reverse) were added by primers. The PCR products were purified and inserted each into the </ins>[<ins class="diffchange diffchange-inline">http</ins>://<ins class="diffchange diffchange-inline">parts</ins>.igem.org/<ins class="diffchange diffchange-inline">Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct] by Golden Gate assembly.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">For the constructs with the linkers sgt2, may1, ord1, ord3, sho1, sho2 and flex, a cloning intermediate containing both lambda lysozyme and the selection marker ccdB instead of the mRFP selection marker was created by Golden Gate assembly of [http</ins>://<ins class="diffchange diffchange-inline">parts.igem.org/Part:BBa_K1362000 NpuDnaE intein RFC(i) circularization construct</ins>] <ins class="diffchange diffchange-inline">and two inserts with subsequent [REF TO MAT/MET religation]. The first insert was created by a PCR of the coding sequence of lambda lysozyme without start and stop codon aquired from genomic lambda DNA. Overhangs containing a BsaI site, the RFC[i] E standard overhang, the coding sequences of the extein CWE (forward) and a BsaI site (reverse) were added by primers. The ccdB insert was created by PCR of the ccdB selection marker from pDONR. Overhangs containing a BsaI site, the last 4 nucleotides of lambda lysozyme (forward) and the RFC[i] B standard overhang and a BsaI site (reverse) were added by primers.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Inserts coding for the linker sequences with overhangs corresponding to the overhangs caused by BsaI restriction of the cloning intermediate were ordered as DNA-oligos. The linker inserts were annealed and inserted to the cloning intermediate by Golden gate assembly</ins>. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Expression and detection of circular lysozyme variants==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Expression and detection of circular lysozyme variants==</div></td></tr>
</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=360970&oldid=prevBunnech at 22:17, 17 October 20142014-10-17T22:17:32Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 3) Coomassie Gel of the linker constructs| descr=The expression levels of the linker constructs are different. In all lanes you can see intein fragments as result of the trans-splicing reaction.| file=62.png}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 3) Coomassie Gel of the linker constructs| descr=The expression levels of the linker constructs are different. In all lanes you can see intein fragments as result of the trans-splicing reaction.| file=62.png}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 2) Western Blot to prove circularization| descr=To test if lambda lysozyme is circular after the splicing reaction of the intein Npu Dna E we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.| file=<del class="diffchange diffchange-inline">WB_36</del>.jpg}}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 2) Western Blot to prove circularization| descr=To test if lambda lysozyme is circular after the splicing reaction of the intein Npu Dna E we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.| file=<ins class="diffchange diffchange-inline">36_20140923_lysozyme</ins>.jpg}}</div></td></tr>
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</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=352593&oldid=prevBunnech at 20:49, 17 October 20142014-10-17T20:49:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Introduction=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Introduction=</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-quarter| align=right| caption=figure 1) Lambda Lysozyme| descr=Secondary structure of lambda lysozyme. The distance between the two ends is 27.3 <del class="diffchange diffchange-inline">Angströms</del>.| file=lysozyme-pdb.png}}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-quarter| align=right| caption=figure 1) Lambda Lysozyme| descr=Secondary structure of lambda lysozyme. The distance between the two ends is 27.3 <ins class="diffchange diffchange-inline">Ångström</ins>.| file=lysozyme-pdb.png}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Designing linkers for circularization is a non trivial task: Circularization is a narrow path between gaining heat-stability and loosing function due to deformation. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Designing linkers for circularization is a non trivial task: Circularization is a narrow path between gaining heat-stability and loosing function due to deformation. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The choice of the protein to perform the linker screen was constrained by different requirements. First, it needed to be easily and fast expressed in ''E.coli''. We needed to be able to measure its functionality without purification and in an easy, fast, cheap and reliable way. It needed to be known for having a certain stability at high temperature with, at the same time, a loss of functionality so that the heat stabilization could be tested. Finally, the major constrain for the choice was the structure of the protein.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The choice of the protein to perform the linker screen was constrained by different requirements. First, it needed to be easily and fast expressed in ''E.coli''. We needed to be able to measure its functionality without purification and in an easy, fast, cheap and reliable way. It needed to be known for having a certain stability at high temperature with, at the same time, a loss of functionality so that the heat stabilization could be tested. Finally, the major constrain for the choice was the structure of the protein.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>First, we needed a crystal structure of the complete protein at high resolution. Second, the ends should be separated by a distance of 15 to 30 <del class="diffchange diffchange-inline">Angströms</del>, so that the rigid linkers containing alpha helices would be relevant. On the other hand, the linker should not pass over the active site of the protein. Furthermore it should be easy to obtain. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>First, we needed a crystal structure of the complete protein at high resolution. Second, the ends should be separated by a distance of 15 to 30 <ins class="diffchange diffchange-inline">Ångström</ins>, so that the rigid linkers containing alpha helices would be relevant. On the other hand, the linker should not pass over the active site of the protein. Furthermore it should be easy to obtain. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Lysozymes are well characterized enzymes that are able to digest the peptidoglycans that form the bacterial wall. This process is performed by many different species for different applications, including antibacterial defence by plants and animals [[#References|[1]]] or bacterial penetration by viruses [[#References|[2]]]. On top, lysozyme is applied in different fields of biotechnology and medicine. It is notably one of the most important proteins in food preservation and is produced in the 100 tons scale a year. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Lysozymes are well characterized enzymes that are able to digest the peptidoglycans that form the bacterial wall. This process is performed by many different species for different applications, including antibacterial defence by plants and animals [[#References|[1]]] or bacterial penetration by viruses [[#References|[2]]]. On top, lysozyme is applied in different fields of biotechnology and medicine. It is notably one of the most important proteins in food preservation and is produced in the 100 tons scale a year. </div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=324942&oldid=prevMaxW at 14:42, 17 October 20142014-10-17T14:42:08Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 3) Coomassie Gel of the linker constructs| descr=The expression levels of the linker constructs are different. In all lanes you can see intein fragments as result of the trans-splicing reaction.| file=62.png}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 3) Coomassie Gel of the linker constructs| descr=The expression levels of the linker constructs are different. In all lanes you can see intein fragments as result of the trans-splicing reaction.| file=62.png}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 2) Western Blot to <del class="diffchange diffchange-inline">proof </del>circularization| descr=To test if lambda lysozyme is circular after the splicing reaction of the intein Npu Dna E we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.| file=WB_36.jpg}}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/templates/image-half| align=right| caption=Figure 2) Western Blot to <ins class="diffchange diffchange-inline">prove </ins>circularization| descr=To test if lambda lysozyme is circular after the splicing reaction of the intein Npu Dna E we did a Western Blot with Penta·His Antibody. There is a clear shift between linear and circular samples.| file=WB_36.jpg}}</div></td></tr>
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</table>MaxWhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=322746&oldid=prevBunnech: /* The long way to setup the assay */2014-10-17T14:00:27Z<p><span class="autocomment">The long way to setup the assay</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Aim of this assay was to see differences in the temperature stability of the different lysozyme constructs, linear and circular, after a heat shock of half of an hour between 35°C and 55°C. The circular lysozyme constructs are clearly working and the probes with the rigid linker work better than the construct including a His-tag. Before declaring a difference between circular and linear constructs we needed more results for great statistics.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Aim of this assay was to see differences in the temperature stability of the different lysozyme constructs, linear and circular, after a heat shock of half of an hour between 35°C and 55°C. The circular lysozyme constructs are clearly working and the probes with the rigid linker work better than the construct including a His-tag. Before declaring a difference between circular and linear constructs we needed more results for great statistics.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>====10th of September====</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>====10th of September <ins class="diffchange diffchange-inline">(1)</ins>====</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next to the first assay we tested a second assay based on an increase of fluorescence intensity. We performed the assay with many different dilutions of substrate and of lysozymes, testing the fluorescein labeled peptidoglycane.To proof the labeling we added the supernatants of the washing steps to see whether they are still fluorescing or not. The fluorescence absolutely doesn't behave as we expected. We don't see any enlargement of the fluorescence. The different curves have huge variations between them.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next to the first assay we tested a second assay based on an increase of fluorescence intensity. We performed the assay with many different dilutions of substrate and of lysozymes, testing the fluorescein labeled peptidoglycane.To proof the labeling we added the supernatants of the washing steps to see whether they are still fluorescing or not. The fluorescence absolutely doesn't behave as we expected. We don't see any enlargement of the fluorescence. The different curves have huge variations between them.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>====10th of September====</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>====10th of September <ins class="diffchange diffchange-inline">(2)</ins>====</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To have a higher statistical coverage we reproduced the last assays. The temperature span of the heat shock was 37°C until 54°C. From this assay on, we could test and evaluate different linkertypes properly, as we have introduced a fitting method, showing the activity of the lysozymes as the exponent of the exponential decay. As we always had different expression levels, we introduced a normalization of the activity to the activity at 37°C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To have a higher statistical coverage we reproduced the last assays. The temperature span of the heat shock was 37°C until 54°C. From this assay on, we could test and evaluate different linkertypes properly, as we have introduced a fitting method, showing the activity of the lysozymes as the exponent of the exponential decay. As we always had different expression levels, we introduced a normalization of the activity to the activity at 37°C.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>There seems to exist some offset activity, the lysozymes always keep.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>There seems to exist some offset activity, the lysozymes always keep.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The final protocol you can find in in Materials and Methods or in parts of the text above!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The final protocol you can find in in Materials and Methods or in parts of the text above!</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</div></td></tr>
</table>Bunnechhttp://2014.igem.org/wiki/index.php?title=Team:Heidelberg/pages/Linker_Screening&diff=322587&oldid=prevBunnech: /* Results */2014-10-17T13:57:14Z<p><span class="autocomment">Results</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We could show, that the data was well described by applying product inhibited Michaelis Menten kinetics. Further on we found out, that the model's quality did not increase significantly by letting the enzyme kinetics ($K_M, K_I$) parameters change over temperature. This was done by making identifiability analysis of model parameters. We showed, that when not restraining $K_M$ and $K_I$ for different temperatures none of the important parameters was identifiable anymore. For better readability we introduced parameters representing the loss in activity after heat shock. These were called activities at a temperature and were built as the ratio between $V^T_{Max}$ and $V^{37°C}_{Max}$. These parameters were the most interesting ones for us. When each lysozyme was modeled with constant $K_M$ and $K_I$ for different temperatures nearly the activities were identifiable within 95% confidence level. For some very good datasets even the kinetic parameters $K_M$ and $K_I$ could be identified on a lower confidence level. Therefore we concluded that the change in these parameters for the different temperatures was minor and could be neglected in the further evaluation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We could show, that the data was well described by applying product inhibited Michaelis Menten kinetics. Further on we found out, that the model's quality did not increase significantly by letting the enzyme kinetics ($K_M, K_I$) parameters change over temperature. This was done by making identifiability analysis of model parameters. We showed, that when not restraining $K_M$ and $K_I$ for different temperatures none of the important parameters was identifiable anymore. For better readability we introduced parameters representing the loss in activity after heat shock. These were called activities at a temperature and were built as the ratio between $V^T_{Max}$ and $V^{37°C}_{Max}$. These parameters were the most interesting ones for us. When each lysozyme was modeled with constant $K_M$ and $K_I$ for different temperatures nearly the activities were identifiable within 95% confidence level. For some very good datasets even the kinetic parameters $K_M$ and $K_I$ could be identified on a lower confidence level. Therefore we concluded that the change in these parameters for the different temperatures was minor and could be neglected in the further evaluation.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We showed that the different linker constructs show different changes in activity after heat shock, figure <del class="diffchange diffchange-inline">5</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We showed that the different linker constructs show different changes in activity after heat shock, figure <ins class="diffchange diffchange-inline">7</ins>. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>caption=Figure <del class="diffchange diffchange-inline">5</del>, Activity after heatshock|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>caption=Figure <ins class="diffchange diffchange-inline">7</ins>, Activity after heatshock|</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The errors are estimations from the standard deviations of the different replicates. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>descr=Ratio of activity after a heatshock of a certain temperature. The errors are estimations from the standard deviations of the different replicates. A clear difference can be seen for ord1- and ord3-linker in comparison to the flexible linker and the linear lysozyme|</div></td></tr>
</table>Bunnech