http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Boo2012.igem.org - User contributions [en]2024-03-28T15:54:13ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-27T00:56:08Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
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<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-27T00:05:21Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
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<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
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==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
<br />
'''Aim:''' To determine the ability of BBa_K729019 to act as a biosafety device.<br />
<br />
<br />
'''Method:''' We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
'''Results:''' As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
<br />
'''Conclusion:''' We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
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== 17-3==<br />
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<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | First Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0|| 0.001|| 0.001|| 0.001 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 ||0.003 ||0.003 ||0.004<br />
|-<br />
|2|| 0.005|| 0.008|| 0.005 ||0.009 ||0.005 ||0.008<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009 ||0.02 ||0.014 ||0.018<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024 ||0.03 ||0.026 ||0.028<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 ||0.064 ||0.057 ||0.066<br />
|-<br />
|6|| 0.084|| 0.116|| 0.086 ||0.08 ||0.088 ||0.89<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189 ||0.18 ||0.188 ||0.179<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303 ||0.306 ||0.297 ||0.292<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 ||0.667 ||0.678 ||0.669<br />
|-<br />
|10|| 0.801|| 1.499|| 0.807 ||0.787 ||0.799 ||0.777<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92 ||0.921 ||0.955 ||0.969<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034 ||1.079 ||1.111 ||1.121<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Second Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.001 ||0.001 ||0.001 ||0.001 ||0.001 ||0.002<br />
|-<br />
|1||0.004 ||0.006 ||0.006 ||0.003 ||0.002 ||0.004<br />
|-<br />
|2||0.006 ||0.009 ||0.008 ||0.008 ||0.006 ||0.01<br />
|-<br />
|3||0.009 ||0.014 ||0.011 ||0.026 ||0.017 ||0.023<br />
|-<br />
|4||0.028 ||0.029 ||0.028 ||0.034 ||0.029 ||0.034<br />
|-<br />
|5||0.048 ||0.07 ||0.066 ||0.068 ||0.066 ||0.072<br />
|-<br />
|6||0.079 ||0.111 ||0.107 ||0.092 ||0.096 ||0.099<br />
|-<br />
|7||0.181 ||0.207 ||0.203 ||0.179 ||0.185 ||0.191<br />
|-<br />
|8||0.28 ||0.47 ||0.36 ||0.311 ||0.289 ||0.298<br />
|-<br />
|9||0.689 ||0.919 ||0.8 ||0.671 ||0.672 ||0.683<br />
|-<br />
|10||0.798 ||1.491 ||1.299 ||0.781 ||0.788 ||0.795<br />
|-<br />
|11||0.909 ||1.8 ||1.664 ||0.924 ||0.974 ||0.981<br />
|-<br />
|12||1.018 ||2.038 ||1.892 ||1.073 ||1.083 ||1.097<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Third Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.002 ||0.001 ||0.002 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1||0.005 ||0.003 ||0.005 ||0.002 ||0.004 ||0.003<br />
|-<br />
|2||0.007 ||0.006 ||0.007 ||0.007 ||0.005 ||0.006<br />
|-<br />
|3||0.011 ||0.016 ||0.01 ||0.018 ||0.014 ||0.017<br />
|-<br />
|4||0.029 ||0.033 ||0.027 ||0.027 ||0.023 ||0.025<br />
|-<br />
|5||0.05 ||0.08 ||0.049 ||0.063 ||0.051 ||0.057<br />
|-<br />
|6||0.087 ||0.116 ||0.085 ||0.091 ||0.083 ||0.081<br />
|-<br />
|7||0.185 ||0.211 ||0.187 ||0.17 ||0.184 ||0.178<br />
|-<br />
|8||0.309 ||0.481 ||0.306 ||0.291 ||0.297 ||0.29<br />
|-<br />
|9||0.681 ||0.924 ||0.799 ||0.666 ||0.686 ||0.68<br />
|-<br />
|10||0.806 ||1.502 ||0.987 ||0.773 ||0.79 ||0.782<br />
|-<br />
|11||0.917 ||1.811 ||0.969 ||0.914 ||0.95 ||0.941<br />
|-<br />
|12||1.031 ||2.049 ||1.159 ||1.064 ||1.099 ||1.093<br />
|-<br />
|}<br />
<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
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</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-27T00:04:30Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
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<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
<br />
'''Aim:''' To determine the ability of BBa_K729019 to act as a biosafety device.<br />
<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
'''Results:''' As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
<br />
'''Conclusion:''' We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
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</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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<br />
== 17-3==<br />
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<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | First Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0|| 0.001|| 0.001|| 0.001 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 ||0.003 ||0.003 ||0.004<br />
|-<br />
|2|| 0.005|| 0.008|| 0.005 ||0.009 ||0.005 ||0.008<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009 ||0.02 ||0.014 ||0.018<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024 ||0.03 ||0.026 ||0.028<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 ||0.064 ||0.057 ||0.066<br />
|-<br />
|6|| 0.084|| 0.116|| 0.086 ||0.08 ||0.088 ||0.89<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189 ||0.18 ||0.188 ||0.179<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303 ||0.306 ||0.297 ||0.292<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 ||0.667 ||0.678 ||0.669<br />
|-<br />
|10|| 0.801|| 1.499|| 0.807 ||0.787 ||0.799 ||0.777<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92 ||0.921 ||0.955 ||0.969<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034 ||1.079 ||1.111 ||1.121<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Second Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.001 ||0.001 ||0.001 ||0.001 ||0.001 ||0.002<br />
|-<br />
|1||0.004 ||0.006 ||0.006 ||0.003 ||0.002 ||0.004<br />
|-<br />
|2||0.006 ||0.009 ||0.008 ||0.008 ||0.006 ||0.01<br />
|-<br />
|3||0.009 ||0.014 ||0.011 ||0.026 ||0.017 ||0.023<br />
|-<br />
|4||0.028 ||0.029 ||0.028 ||0.034 ||0.029 ||0.034<br />
|-<br />
|5||0.048 ||0.07 ||0.066 ||0.068 ||0.066 ||0.072<br />
|-<br />
|6||0.079 ||0.111 ||0.107 ||0.092 ||0.096 ||0.099<br />
|-<br />
|7||0.181 ||0.207 ||0.203 ||0.179 ||0.185 ||0.191<br />
|-<br />
|8||0.28 ||0.47 ||0.36 ||0.311 ||0.289 ||0.298<br />
|-<br />
|9||0.689 ||0.919 ||0.8 ||0.671 ||0.672 ||0.683<br />
|-<br />
|10||0.798 ||1.491 ||1.299 ||0.781 ||0.788 ||0.795<br />
|-<br />
|11||0.909 ||1.8 ||1.664 ||0.924 ||0.974 ||0.981<br />
|-<br />
|12||1.018 ||2.038 ||1.892 ||1.073 ||1.083 ||1.097<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Third Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.002 ||0.001 ||0.002 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1||0.005 ||0.003 ||0.005 ||0.002 ||0.004 ||0.003<br />
|-<br />
|2||0.007 ||0.006 ||0.007 ||0.007 ||0.005 ||0.006<br />
|-<br />
|3||0.011 ||0.016 ||0.01 ||0.018 ||0.014 ||0.017<br />
|-<br />
|4||0.029 ||0.033 ||0.027 ||0.027 ||0.023 ||0.025<br />
|-<br />
|5||0.05 ||0.08 ||0.049 ||0.063 ||0.051 ||0.057<br />
|-<br />
|6||0.087 ||0.116 ||0.085 ||0.091 ||0.083 ||0.081<br />
|-<br />
|7||0.185 ||0.211 ||0.187 ||0.17 ||0.184 ||0.178<br />
|-<br />
|8||0.309 ||0.481 ||0.306 ||0.291 ||0.297 ||0.29<br />
|-<br />
|9||0.681 ||0.924 ||0.799 ||0.666 ||0.686 ||0.68<br />
|-<br />
|10||0.806 ||1.502 ||0.987 ||0.773 ||0.79 ||0.782<br />
|-<br />
|11||0.917 ||1.811 ||0.969 ||0.914 ||0.95 ||0.941<br />
|-<br />
|12||1.031 ||2.049 ||1.159 ||1.064 ||1.099 ||1.093<br />
|-<br />
|}<br />
<br />
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<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
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</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-27T00:03:41Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
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<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
<br />
'''Aim:''' To determine the ability of BBa_K729019 to act as a biosafety device by preventing transformation of competent cells with material from cells harbouring the biobrick. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
'''Results:''' As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
<br />
'''Conclusion:''' We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
<br />
== 17-3==<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | First Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0|| 0.001|| 0.001|| 0.001 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 ||0.003 ||0.003 ||0.004<br />
|-<br />
|2|| 0.005|| 0.008|| 0.005 ||0.009 ||0.005 ||0.008<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009 ||0.02 ||0.014 ||0.018<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024 ||0.03 ||0.026 ||0.028<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 ||0.064 ||0.057 ||0.066<br />
|-<br />
|6|| 0.084|| 0.116|| 0.086 ||0.08 ||0.088 ||0.89<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189 ||0.18 ||0.188 ||0.179<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303 ||0.306 ||0.297 ||0.292<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 ||0.667 ||0.678 ||0.669<br />
|-<br />
|10|| 0.801|| 1.499|| 0.807 ||0.787 ||0.799 ||0.777<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92 ||0.921 ||0.955 ||0.969<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034 ||1.079 ||1.111 ||1.121<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Second Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.001 ||0.001 ||0.001 ||0.001 ||0.001 ||0.002<br />
|-<br />
|1||0.004 ||0.006 ||0.006 ||0.003 ||0.002 ||0.004<br />
|-<br />
|2||0.006 ||0.009 ||0.008 ||0.008 ||0.006 ||0.01<br />
|-<br />
|3||0.009 ||0.014 ||0.011 ||0.026 ||0.017 ||0.023<br />
|-<br />
|4||0.028 ||0.029 ||0.028 ||0.034 ||0.029 ||0.034<br />
|-<br />
|5||0.048 ||0.07 ||0.066 ||0.068 ||0.066 ||0.072<br />
|-<br />
|6||0.079 ||0.111 ||0.107 ||0.092 ||0.096 ||0.099<br />
|-<br />
|7||0.181 ||0.207 ||0.203 ||0.179 ||0.185 ||0.191<br />
|-<br />
|8||0.28 ||0.47 ||0.36 ||0.311 ||0.289 ||0.298<br />
|-<br />
|9||0.689 ||0.919 ||0.8 ||0.671 ||0.672 ||0.683<br />
|-<br />
|10||0.798 ||1.491 ||1.299 ||0.781 ||0.788 ||0.795<br />
|-<br />
|11||0.909 ||1.8 ||1.664 ||0.924 ||0.974 ||0.981<br />
|-<br />
|12||1.018 ||2.038 ||1.892 ||1.073 ||1.083 ||1.097<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Third Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.002 ||0.001 ||0.002 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1||0.005 ||0.003 ||0.005 ||0.002 ||0.004 ||0.003<br />
|-<br />
|2||0.007 ||0.006 ||0.007 ||0.007 ||0.005 ||0.006<br />
|-<br />
|3||0.011 ||0.016 ||0.01 ||0.018 ||0.014 ||0.017<br />
|-<br />
|4||0.029 ||0.033 ||0.027 ||0.027 ||0.023 ||0.025<br />
|-<br />
|5||0.05 ||0.08 ||0.049 ||0.063 ||0.051 ||0.057<br />
|-<br />
|6||0.087 ||0.116 ||0.085 ||0.091 ||0.083 ||0.081<br />
|-<br />
|7||0.185 ||0.211 ||0.187 ||0.17 ||0.184 ||0.178<br />
|-<br />
|8||0.309 ||0.481 ||0.306 ||0.291 ||0.297 ||0.29<br />
|-<br />
|9||0.681 ||0.924 ||0.799 ||0.666 ||0.686 ||0.68<br />
|-<br />
|10||0.806 ||1.502 ||0.987 ||0.773 ||0.79 ||0.782<br />
|-<br />
|11||0.917 ||1.811 ||0.969 ||0.914 ||0.95 ||0.941<br />
|-<br />
|12||1.031 ||2.049 ||1.159 ||1.064 ||1.099 ||1.093<br />
|-<br />
|}<br />
<br />
<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-27T00:03:05Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
<br />
'''<br />
Aim:''' To determine the ability of BBa_K729019 to act as a biosafety device by preventing transformation of competent cells with material from cells harbouring the biobrick. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
'''Results:'''<br />
<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
<br />
'''Conclusion:'''<br />
<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
<br />
== 17-3==<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | First Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0|| 0.001|| 0.001|| 0.001 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 ||0.003 ||0.003 ||0.004<br />
|-<br />
|2|| 0.005|| 0.008|| 0.005 ||0.009 ||0.005 ||0.008<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009 ||0.02 ||0.014 ||0.018<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024 ||0.03 ||0.026 ||0.028<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 ||0.064 ||0.057 ||0.066<br />
|-<br />
|6|| 0.084|| 0.116|| 0.086 ||0.08 ||0.088 ||0.89<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189 ||0.18 ||0.188 ||0.179<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303 ||0.306 ||0.297 ||0.292<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 ||0.667 ||0.678 ||0.669<br />
|-<br />
|10|| 0.801|| 1.499|| 0.807 ||0.787 ||0.799 ||0.777<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92 ||0.921 ||0.955 ||0.969<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034 ||1.079 ||1.111 ||1.121<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Second Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.001 ||0.001 ||0.001 ||0.001 ||0.001 ||0.002<br />
|-<br />
|1||0.004 ||0.006 ||0.006 ||0.003 ||0.002 ||0.004<br />
|-<br />
|2||0.006 ||0.009 ||0.008 ||0.008 ||0.006 ||0.01<br />
|-<br />
|3||0.009 ||0.014 ||0.011 ||0.026 ||0.017 ||0.023<br />
|-<br />
|4||0.028 ||0.029 ||0.028 ||0.034 ||0.029 ||0.034<br />
|-<br />
|5||0.048 ||0.07 ||0.066 ||0.068 ||0.066 ||0.072<br />
|-<br />
|6||0.079 ||0.111 ||0.107 ||0.092 ||0.096 ||0.099<br />
|-<br />
|7||0.181 ||0.207 ||0.203 ||0.179 ||0.185 ||0.191<br />
|-<br />
|8||0.28 ||0.47 ||0.36 ||0.311 ||0.289 ||0.298<br />
|-<br />
|9||0.689 ||0.919 ||0.8 ||0.671 ||0.672 ||0.683<br />
|-<br />
|10||0.798 ||1.491 ||1.299 ||0.781 ||0.788 ||0.795<br />
|-<br />
|11||0.909 ||1.8 ||1.664 ||0.924 ||0.974 ||0.981<br />
|-<br />
|12||1.018 ||2.038 ||1.892 ||1.073 ||1.083 ||1.097<br />
|-<br />
|}<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! colspan="7" style="background: #efefef;" | Third Inocculation<br />
|- <br />
! x <br />
! colspan="3" | Nuclease positive <br />
! colspan="3" | Nuclease negative (control) <br />
|-<br />
|Time || 1|| 2|| 3 || 1|| 2|| 3<br />
|-<br />
|0||0.002 ||0.001 ||0.002 ||0.001 ||0.001 ||0.001<br />
|-<br />
|1||0.005 ||0.003 ||0.005 ||0.002 ||0.004 ||0.003<br />
|-<br />
|2||0.007 ||0.006 ||0.007 ||0.007 ||0.005 ||0.006<br />
|-<br />
|3||0.011 ||0.016 ||0.01 ||0.018 ||0.014 ||0.017<br />
|-<br />
|4||0.029 ||0.033 ||0.027 ||0.027 ||0.023 ||0.025<br />
|-<br />
|5||0.05 ||0.08 ||0.049 ||0.063 ||0.051 ||0.057<br />
|-<br />
|6||0.087 ||0.116 ||0.085 ||0.091 ||0.083 ||0.081<br />
|-<br />
|7||0.185 ||0.211 ||0.187 ||0.17 ||0.184 ||0.178<br />
|-<br />
|8||0.309 ||0.481 ||0.306 ||0.291 ||0.297 ||0.29<br />
|-<br />
|9||0.681 ||0.924 ||0.799 ||0.666 ||0.686 ||0.68<br />
|-<br />
|10||0.806 ||1.502 ||0.987 ||0.773 ||0.79 ||0.782<br />
|-<br />
|11||0.917 ||1.811 ||0.969 ||0.914 ||0.95 ||0.941<br />
|-<br />
|12||1.031 ||2.049 ||1.159 ||1.064 ||1.099 ||1.093<br />
|-<br />
|}<br />
<br />
<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:57:35Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/File:Data_13.pngFile:Data 13.png2012-10-26T23:56:57Z<p>Boo: </p>
<hr />
<div></div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:56:50Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
<br />
<br />
<br />
[[File:Data 13.png|700px|center]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:51:26Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
<br />
<br />
<br />
[[File:Data 13Bouran-So.png|700px|center]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:51:11Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
<br />
<br />
[[File:Data 13Bouran-So.png|700px|center]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:50:43Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
[[File:Data 13Bouran-So.png|600px|center]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:49:50Z<p>Boo: /* IrrE enable E. coli to survive in marine environments */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
[[File:Data 13Bouran-So.png]]<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/File:Data_13Bouran-So.pngFile:Data 13Bouran-So.png2012-10-26T23:49:27Z<p>Boo: </p>
<hr />
<div></div>Boohttp://2012.igem.org/Team:University_College_London/Module_5/ResultsTeam:University College London/Module 5/Results2012-10-26T23:46:31Z<p>Boo: /* Characterisation of BBa_K398108 for Comparison */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 5: Salt Tolerance=<br />
{{:Team:University_College_London/templates/module5menu}}<br />
<br />
== Characterisation of BBa_K729005 (IrrE) ==<br />
<br />
From our results, we observed that ''E. coli'' transformed with the IrrE gene exhibits greater salt tolerance than their non-transformed counterparts. This is revealed in the greater optical density (OD) the cells grow to in high salt media, as well as their increased growth rates in the exponential growth phase. Our results are consistent with the results obtained in the original <span class="footnote" title="Pan">paper</span>, establishing the fact that our cells are indeed expressing the IrrE global regulator.<br />
<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_Growth_Rate_%_Increase.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_LB1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.3M_NaCl1.png|475px]]<br />
[[File:UniversityCollegeLondon_IrrE_0.6M_NaCl1.png|475px|centre]]<br />
<br />
== IrrE enable E. coli to survive in marine environments ==<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Characterisation of BBa_K398108 for Comparison ==<br />
<br />
<br />
We have observed that the results we have obtained for the characterisation of BBa_K398108 are consistent with those of the TU Delft '10 iGEM team. From the growth curves obtained (see graph below), an increase growth rate during the exponential phase is observed in ''E. Coli'' expressing BBa_K398108 as opposed to the wild type when the salt concentration of the media is elevated.<br />
<br />
However, while we have managed to replicate the results of the TU Delft '10 iGEM team, we question the viability of this BioBrick for conferring salt tolerance in ''E. coli''. While the growth rate is improved for the cells expressing the BioBrick, the overall cell density is not - from our results, the final OD of the cells in the stationary phase is not higher than that of the wild-type.<br />
<br />
Examining the <span class="footnote" title="Pan">literature</span>, a better gauge of salt tolerance can be found via an increase in OD over the wild type cells in increased salt concentrations, which this BioBrick has not been shown to do. As such, the choice to use K398108 to confer salt tolerance on our cells would remain questionable at best.<br />
<br />
[[File:UniversityCollegeLondon Salt Tolerance K398108 Growth Curve.png|470px]]<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_K398108_Growth_rate.png|470px]]<br />
<br />
As the only available evidence for the function of the TU Delft ’10 Salt Tolerance BioBrick (K398108) comes from analysis of the growth rate during the exponential phase, this is used to draw direct performance comparison with the UCL ’12 BioBrick (K729005) (NB: TU Delft '10 data approximate and based on info taken from the team's wiki) which it has already been shown provides this role. The plot below highlights the impressive performance advantage that the UCL construct has over the previous BioBrick for enduring a high salinity environment.<br />
<br />
[[File:UniversityCollegeLondon_Salt_Tolerance_Growth_Comp.png|650px|centre]]</div>Boohttp://2012.igem.org/File:Data_13-1.jpgFile:Data 13-1.jpg2012-10-26T23:40:06Z<p>Boo: </p>
<hr />
<div></div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T23:35:53Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
<br />
== 17-3==<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
| '''First innoculation''' !! !! !! <br />
|- <br />
| Nuclease positive !! !! !! <br />
|-<br />
|Time !! 1!! 2!! 3 <br />
|-<br />
|0|| 0.001|| 0.001|| 0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 <br />
|-<br />
|2|| 0.005|| 0.008|| 0.005<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 <br />
|-<br />
|6|| 0.084|| 0.116|| 0.086<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 <br />
|-<br />
|10|| 0.801|| 1.499|| 0.807<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034<br />
|-<br />
|}<br />
<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T23:35:36Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
<br />
== 17-3==<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
| '''First innoculation''' !! !! !! <br />
|- <br />
| Nuclease positive !! !! !! <br />
|-<br />
|Time !! 1!! 2!! 3 <br />
|-<br />
|0 0.001|| 0.001|| 0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 <br />
|-<br />
|2|| 0.005|| 0.008|| 0.005<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 <br />
|-<br />
|6|| 0.084|| 0.116|| 0.086<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 <br />
|-<br />
|10|| 0.801|| 1.499|| 0.807<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034<br />
|-<br />
|}<br />
<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T23:35:15Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
<br />
== 17-3==<br />
<br />
<br />
<br />
<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
| '''First innoculation''' !! !! !! <br />
|- <br />
| Nuclease positive !! !! !! <br />
|-<br />
|Time !! 1!! 2!! 3 <br />
|-<br />
|0 0.001|| 0.001|| 0.001<br />
|-<br />
|1|| 0.003|| 0.005|| 0.004 <br />
|-<br />
|2|| 0.005|| 0.008|| 0.005<br />
|-<br />
|3|| 0.008|| 0.011|| 0.009<br />
|-<br />
|4|| 0.021|| 0.03|| 0.024<br />
|-<br />
|5|| 0.053|| 0.076|| 0.059 <br />
|-<br />
|6|| 0.084|| 0.116|| 0.086<br />
|-<br />
|7|| 0.183|| 0.217|| 0.189<br />
|-<br />
|8|| 0.299|| 0.479|| 0.303<br />
|-<br />
|9|| 0.684|| 0.918|| 0.69 <br />
|-<br />
|10|| 0.801|| 1.499|| 0.807<br />
|-<br />
|11|| 0.913 ||1.804|| 0.92<br />
|-<br />
|12|| 1.022 ||2.033|| 1.034<br />
|-<br />
|}<br />
<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
<br />
== 17-4==<br />
<br />
<br />
{| class="bigtable"<br />
|-<br />
! Time!! Run 1 !! Run 2 !! Run 3<br />
|-<br />
| 0 ||0.001 || 0.001|| 0.001<br />
|-<br />
| 1 || 0.002 || 0.003||0.004<br />
|-<br />
| 2 || 0.008|| 0.007 || 0.008<br />
|-<br />
| 3 || 0.02 || 0.019|| 0.019<br />
|-<br />
| 4 || 0.029 || 0.026 ||0.027<br />
|-<br />
| 5 || 0.06|| 0.059 || 0.062<br />
|-<br />
| 6 || 0.087|| 0.089|| 0.089<br />
|-<br />
| 7 || 0.184|| 0.185 || 0.183<br />
|-<br />
| 8 || 0.302|| 0.297 || 0.292<br />
|-<br />
| 9 || 0.669|| 0.678 || 0.675<br />
|}<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T23:34:18Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729019) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig 1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig 1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig 1). BBa_K729019 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T22:18:10Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig 1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig 1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig 1). BBa_K729019 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T22:16:54Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
== 17-3==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
</html><br />
== 17-4==<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ConclusionTeam:University College London/Module 6/Conclusion2012-10-26T22:15:04Z<p>Boo: /* Conclusion */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Conclusion ==<br />
<br />
We have successfully transformed our cells to produce a periplasmic nuclease, a necessary feature to prevent transformation of wild-type cells in the marine environment. Furthermore, we have proven that this BioBrick does not adversely affect the viability of our cells, allowing it to be utilised as part of a containment system for engineered cells. This also demonstrates that DsbA signal works otherwise the nuclease would have caused cell death as cytosolic nucleases are lethal and more commonly used as kill-swithces<br />
<br />
Our DNAse assay is helpful not only for identifying the activity of our nuclease but also this is a nice system to quickly show if DsbA-mediated periplasm trafficking is possible.<br />
<br />
'''We successfully demonstarte that material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells. This suggests periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.''' <br />
<br />
In addition to this we are able to predict nuclease activity which correlates to the colony mass. This shows that containment model is realistic.<br />
<br />
Future work would attempt to construct a three-fold containment system, in order to create a more robust system capable of prevent other mechanisms of horizontal gene transfer, such as transduction and bacterial conjugation.</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ConclusionTeam:University College London/Module 6/Conclusion2012-10-26T22:14:35Z<p>Boo: /* Conclusion */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Conclusion ==<br />
<br />
We have successfully transformed our cells to produce a periplasmic nuclease, a necessary feature to prevent transformation of wild-type cells in the marine environment. Furthermore, we have proven that this BioBrick does not adversely affect the viability of our cells, allowing it to be utilised as part of a containment system for engineered cells. This also demonstrates that DsbA signal works otherwise the nuclease would have caused cell death as cytosolic nucleases are lethal and more commonly used as kill-swithces<br />
<br />
Our DNAse assay is helpful not only for identifying the activity of our nuclease but also this is a nice system to quickly show if DsbA-mediated periplasm trafficking is possible.<br />
<br />
'''We successfully demonstarte that material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells. This suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.''' <br />
<br />
In addition to this we are able to predict nuclease activity which correlates to the colony mass. This shows that containment model is realistic.<br />
<br />
Future work would attempt to construct a three-fold containment system, in order to create a more robust system capable of prevent other mechanisms of horizontal gene transfer, such as transduction and bacterial conjugation.</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T22:10:40Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
Furthermore, we show that '''material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells'''. As such, periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T22:09:09Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
We further show that '''material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells'''. We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:28:04Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with '''W3110:''' unmodified E. coli W3110 disruptate, '''Laccase''': W3110 harbouring the BBa_K729006 laccase plasmid and '''Nuclease''': E. coli W3110 harbouring the BBa_K729019 nuclease plasmid. ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig 1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig 1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig 1). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:26:50Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|'''Figure 1.''' Transformation of comercially competent TOP10 E. Coli cells with unmodified E. coli W3110 disruptate, Laccase:W3110 harbouring the BBa_K729006 laccase plasmid and Nuclease: E. coli W3110 harbouring the BBa_K729019 nuclease plasmid ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig1). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:26:30Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=|Figure 1. Transformation of comercially competent TOP10 E. Coli cells with unmodified E. coli W3110 disruptate, Laccase:W3110 harbouring the BBa_K729006 laccase plasmid and Nuclease: E. coli W3110 harbouring the BBa_K729019 nuclease plasmid ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig1). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:25:40Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt=Figure 1. Transformation of comercially competent TOP10 E. Coli cells with unmodified E. coli W3110 disruptate, Laccase:W3110 harbouring the BBa_K729006 laccase plasmid and Nuclease: E. coli W3110 harbouring the BBa_K729019 nuclease plasmid ]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig1). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:23:14Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|thumb|center|600px|alt= Figure 1.]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (Fig1). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (Fig1), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (Fig1). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T20:15:03Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|600px|center|]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T20:14:33Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg|600px|center|]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
<div class="experimentContent"></html><br />
== 17-3==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
<html><br />
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<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T20:10:38Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
[[File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg]]<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
<html><br />
</div><div class="experiment"></div></html><br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/File:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpgFile:20121025-Bouran.S.NucleaseExpPlates.25.10.12-2.jpg2012-10-26T20:09:41Z<p>Boo: </p>
<hr />
<div></div>Boohttp://2012.igem.org/File:20121025-Bouran.S.NucleaseExpPlates.25.10.12.jpgFile:20121025-Bouran.S.NucleaseExpPlates.25.10.12.jpg2012-10-26T20:03:08Z<p>Boo: </p>
<hr />
<div></div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T18:55:29Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
We further characterise the nuclease Biobrick BBa_K729004/BBa_K729004, showing that '''material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells'''. We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T18:54:18Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
We further characterise the nuclease Biobrick, showing that '''material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells'''. We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T18:54:04Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
We further characterise the nuclease Biobrick, showing that '''material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells'''. We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/CharacterisationTeam:University College London/Module 6/Characterisation2012-10-26T18:53:49Z<p>Boo: /* Characterisation */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== Characterisation ==<br />
<br />
'''"The days of the head-shaking dismissal of the questions concerning risks, and the easy answers referring to the vast experience from the laboratory experiments are over; they have been replaced by scientific investigations of the questions and increased activity in the field of ecology. This is an interesting example of scientific choice being influenced by events in the rest of the society."''' (Molin ''et al.'' 1993)<br />
<br />
In order to determine the activity of the periplasmic nuclease, we will be utilising DNAse agar. This agar contains DNA fragments that would be cleaved by the nuclease, leaving the regions surrounding the colonies free of genetic material. The agar plates can then be stained with a DNA indicator such as Hydrochloric Acid or Toludine Blue, allowing the DNA voids in the agar to be visible.<br />
<br />
<br />
We further characterise the nuclease Biobrick, showing that material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells. We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts. <br />
<br />
<br />
For the toxin/anti-toxin pairs characterisation, we will be transforming our toxic plasmids into cell both with and without the genomic anti-toxin genes. By comparing the ratio of cell viability in each case, we will be able to identify how effective our containment system is. <br />
<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T18:51:04Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
<html><br />
</div><div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
<html></div><br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
<html><br />
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<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T18:50:38Z<p>Boo: /* 17-1 */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
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<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
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<div class="experimentContent"></html><br />
== 17-1==<br />
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<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
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==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
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{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T18:50:15Z<p>Boo: /* 17-1 */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
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{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T18:50:05Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
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<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
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== 17-1==<br />
<html></div><br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
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== 17-2==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
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== 17-4==<br />
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{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/LabBook/Week17Team:University College London/LabBook/Week172012-10-26T18:48:24Z<p>Boo: /* 17-1 */</p>
<hr />
<div>{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index.php?title=Team:University_College_London/js/labbookjs&amp;action=raw&amp;ctype=text/js"></script><br />
</html>{{:Team:University_College_London/templates/labbookmenu}}<html><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" /><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-1.png" /><br />
<div class="experimentContent"></html><br />
== 17-1==<br />
<html></div><br />
<div class="experiment"></div><br />
<img src="http://www.plasticrepublic.org/wikifiles/lab17-2.png"" /><div class="experimentContent"><br />
</html><br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells.<br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells.<br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells.<br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== 17-2==<br />
<html><br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-3.png" /><br />
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== 17-3==<br />
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<img src="http://www.plasticrepublic.org/wikifiles/lab17-4.png"" /><div class="experimentContent"><br />
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== 17-4==<br />
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<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T18:44:57Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_K729006 laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T18:43:59Z<p>Boo: /* Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729019 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_K729006) or the DsbA-Nuclease (BBa_K729019). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_XXXXXX laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729019 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_K729006 only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Module_6/ResultsTeam:University College London/Module 6/Results2012-10-26T18:41:52Z<p>Boo: /* DNase Agar Assay */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
=Module 6: Containment=<br />
{{:Team:University_College_London/templates/module6menu}}<br />
<br />
== DNase Agar Assay ==<br />
The following table shows the results we obtained from the DNase agar test. DNase agar contains DNA, which our periplasmic nuclease digests. This digestion is observed by adding hydrochloric acid to the agar plates, which causes the remaining DNA to precipitate, causing 'cloudiness' to the DNase agar.<br />
<br />
As seen in our DNase agar plate, there is a clear halo surrounding our nuclease transformed cells, indicating a DNA-free zone where the nuclease has digested the DNA in the agar. No such halo is present in our untransformed cells. This indicates that our transformation has been successful, and that BBa_K729004 is working as expected, producing periplasmic nuclease that is capable of digesting extracellular genetic material.<br />
<br />
{| class="wikitable"<br />
|+ DNase Agar Assay<br />
! Control !! BBa_K729019<br />
|- <br />
| [[File:UniversityCollegeLondon_Nuclease_Control.jpg|475px]] || [[File:UniversityCollegeLondon_Nuclease_K729004.jpg|475px]]<br />
|}<br />
<br />
<br />
==Material from cells containing our Biosafety BioBrick is not able to transform commercial competent cells==<br />
<br />
<br />
We demonstrate the ability of a staphylococcal nuclease modified with the DsbA periplasm export signal (BBa_K729004) to prevent transfer of genetically modified material to a commercially competent TOP10 E. coli strain. This observation is consistent with our proposed function of BBa_K729004 to act as a biosafety mechanism preventing uptake of released DNA by naturally competent bacteria. <br />
<br />
We used sonication to disrupt three cell types: unmodified W3110 strain E. coli and W3110 strains harbouring chloramphenical resistance plasmids encoding a laccase (BBa_XXXXXX) or the DsbA-Nuclease (BBa_K729004). All three strains were grown to OD600= 2.0 prior to sonication, in 2mL LB broth which contained 100ug/mL chloramphenical for plasmid-harbouring cells. <br />
<br />
After sonication, disruptates were incubated for 10min at room temperature. 5uL of the disruptate was used to transform an aliquot of TOP10 chemically competent cells, following manufacturer instructions. As a control, we also spread 20uL of the disruptate material directly onto LB agar plates with and without 100ug/mL Chloramphenical. <br />
No colonies were observed on the control plates for any of the three strains (data not shown), indicating sonication had successfully disrupted all cells. <br />
<br />
As expected, transformation of TOP10 cells with disruptate of unmodified W3110 did not yield any cells able to grow on chloramphenicol plates (FigXA). In contrast transformation with disruptate of W3110 harbouring the BBa_XXXXXX laccase plasmid yielded many TOP10 colonies on chloramphenicol plates (FigXA), indicating that plasmid DNA in the disruptate was able to transform TOP10 cells.<br />
<br />
Transformation with disruptate of W3110 harbouring the BBa_K729004 nuclease plasmid yielded no colonies on chloramphenicol plates (FigXC). BBa_K729004 differs from BBa_XXXXXX only in that it contains an ORF encoding the periplasmic nuclease instead of laccase. As such, we conclude the periplasmic nuclease has sufficient DNAase activity in the disruptate to reduce the amount of plasmid DNA present to below the threshold capable of transforming TOP10 cells. <br />
<br />
We suggest periplasmic nuclease expression provides a promising strategy for preventing transfer of genetically modified DNA. This is particularly valuable for the application of synthetic biology in environmental contexts.<br />
<br />
== Colony Halo size Assay ==<br />
<br />
A separate DNase agar assay was also done, in order to determine if there was any correlation between colony size and nuclease production of our nuclease BioBrick (BBa_K729019). DNase agar was plated out with nuclease transformed W3110, at a concentration to produce single colonies on the plates. Colony sizes were measured, as wer the DNA-free zones surrounding the colonies.<br />
<br />
We have determined that a linear correlation exists between colony size and halo diameter, hence suggesting that cells produce nuclease even as they grow.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Colony_Halo_Diameter_correlation.png]]<br />
<br />
== Growth Unaffected by Periplasmic Nuclease Expression ==<br />
<br />
A growth curve assay was done on our nuclease transformed cells in order to determine if there was sufficient intracellular production of nuclease to adversely affect the viability of our cells. The cells were grown in shake flasks, and their optical density at 600nm was measured.<br />
<br />
Our results indicate that the nuclease BioBrick does not negatively affect the growth of our cells. The growth rates obtained from our nuclease transformed cells are comparable to those of the untransformed W3110 cell line.<br />
<br />
[[File:UniversityCollegeLondon_Nuclease_Toxicity_Growth_Curve_Assay.png]]</div>Boohttp://2012.igem.org/Team:University_College_London/Research/MarineBacteriaTeam:University College London/Research/MarineBacteria2012-10-15T14:21:23Z<p>Boo: /* Growth comparison */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
= Marine Chassis =<br />
<br />
==Roseobacter clade bacteria ==<br />
<br />
<br />
<br />
In consultation with Paola R. Gomez-Pereira of the National Oceanography Centre, Southampton, we identified ''Oceanibulbus indolifex'' and ''Roseobacter dentitrificans'' as promising chassis for the expression of our systems. <br />
<br />
The roseobacter clade bacteria, RCB, constitute a significant proportion of coastal and ocean bacterioplankton communities, estimated above 20% and 15% respectively, and are found in a diverse range of marine habitats. (Buchan ''et al.'' 2006). RCB demonstrate numerous traits, including aerobic anoxygenic phototrophy, aromatic compound degradation and recycling of sulphur within the water column and have been implicated in carbon monoxide consumption. (Wagner-Döbler & Biebl, ''et al.'' 2006). Significantly, in the context of plastic Island, they are shown to be major colonisers of submerged surfaces in marine waters (Dang & Lovell ''et al.'' 2002).<br />
<br />
<br />
[[File:UniversityCollegeLondon_O_indolifex_Transformed_Wide.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Medium.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Close.jpg|310px]]<br />
<br />
Figure 1. ''Oceanibulbus indolifex''streaked on marine agar, showing colony morphology.<br />
<br />
==Transformation Protocols== <br />
<br />
Transformation of ''Oceanibulbus indoliflex'' by electroporation. Adapted from (Piekaski ''et al.'' 2009)<br />
<br />
Electrocompetent cells were prepared according to the following protocol: <br />
<br />
45ml Marine Broth was inoculated with 1ml of ''O. indoliflex'' and grown to ~ 0.5 at OD 578. <br />
<br />
10ml volumes were transferred to 50 mL falcons and cells were sedimented for 15mins at 3200 x g in a pre-chilled centrifuge. <br />
<br />
Cells were washed 5 times with 10% (v/v) glycerol, and finally resuspended in 400uL 10% (v/v) glycerol. <br />
<br />
50uL aliquots were made and stored at -80C. <br />
<br />
Electroporation:<br />
<br />
1uL DNA was added to 50uL competent cells to chilled 1mm electrocuvettes, and treated with a field strength of 2.5kV. <br />
<br />
1ml marine broth was added to cells and mixtures were transferred to falcons for incubation overnight at room temperature at 200rpm and finally plated on marine agar supplemented with appropriate antibiotics. Incubated at 30˚C for 2 days.<br />
<br />
==Growth comparison==<br />
<br />
Growth comparison of ''Oceanibulbus indolifex'' and ''Escherichia coli'' in marine and luria broth.<br />
We sought to compare the growth profiles of ''O. indolifex'' and ''E. coli''. <br />
<br />
[[File:UniversityCollegeLondon_Marine_1.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_2.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_3.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_4.png|478px]]<br />
<br />
Figures 2-5. Comparative growth profiles of ''Escherichia coli K-12 substr. W3110'' and ''Oceanibulbus indolifex'' in Marine Broth and LB media at 25°C and 37°C.<br />
<br />
==References==<br />
<br />
1. Brinkhoff, T., Giebel, H.-A., & Simon, M. (2008). Diversity, ecology, and genomics of the Roseobacter clade: a short overview. Archives of microbiology, 189(6), 531–9. <br />
<br />
2. Piekarski, T., Buchholz, I., Drepper, T., Schobert, M., Wagner-Doebler, I., Tielen, P., & Jahn, D. (2009). Genetic tools for the investigation of Roseobacter clade bacteria. BMC microbiology, 9, 265. <br />
<br />
3. Buchan, A., González, J. M., & Moran, M. A. (2005). Overview of the marine roseobacter lineage. Applied and environmental microbiology, 71(10), 5665–77. <br />
<br />
4. Dang, H., & Lovell, C. R. (2002). Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization. Environmental microbiology, 4(5), 287–95. <br />
<br />
5. Wagner-Döbler, I., & Biebl, H. (2006). Environmental biology of the marine Roseobacter lineage. Annual review of microbiology, 60, 255–80. <br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Research/MarineBacteriaTeam:University College London/Research/MarineBacteria2012-10-15T14:20:56Z<p>Boo: /* Roseobacter clade bacteria */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
= Marine Chassis =<br />
<br />
==Roseobacter clade bacteria ==<br />
<br />
<br />
<br />
In consultation with Paola R. Gomez-Pereira of the National Oceanography Centre, Southampton, we identified ''Oceanibulbus indolifex'' and ''Roseobacter dentitrificans'' as promising chassis for the expression of our systems. <br />
<br />
The roseobacter clade bacteria, RCB, constitute a significant proportion of coastal and ocean bacterioplankton communities, estimated above 20% and 15% respectively, and are found in a diverse range of marine habitats. (Buchan ''et al.'' 2006). RCB demonstrate numerous traits, including aerobic anoxygenic phototrophy, aromatic compound degradation and recycling of sulphur within the water column and have been implicated in carbon monoxide consumption. (Wagner-Döbler & Biebl, ''et al.'' 2006). Significantly, in the context of plastic Island, they are shown to be major colonisers of submerged surfaces in marine waters (Dang & Lovell ''et al.'' 2002).<br />
<br />
<br />
[[File:UniversityCollegeLondon_O_indolifex_Transformed_Wide.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Medium.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Close.jpg|310px]]<br />
<br />
Figure 1. ''Oceanibulbus indolifex''streaked on marine agar, showing colony morphology.<br />
<br />
==Transformation Protocols== <br />
<br />
Transformation of ''Oceanibulbus indoliflex'' by electroporation. Adapted from (Piekaski ''et al.'' 2009)<br />
<br />
Electrocompetent cells were prepared according to the following protocol: <br />
<br />
45ml Marine Broth was inoculated with 1ml of ''O. indoliflex'' and grown to ~ 0.5 at OD 578. <br />
<br />
10ml volumes were transferred to 50 mL falcons and cells were sedimented for 15mins at 3200 x g in a pre-chilled centrifuge. <br />
<br />
Cells were washed 5 times with 10% (v/v) glycerol, and finally resuspended in 400uL 10% (v/v) glycerol. <br />
<br />
50uL aliquots were made and stored at -80C. <br />
<br />
Electroporation:<br />
<br />
1uL DNA was added to 50uL competent cells to chilled 1mm electrocuvettes, and treated with a field strength of 2.5kV. <br />
<br />
1ml marine broth was added to cells and mixtures were transferred to falcons for incubation overnight at room temperature at 200rpm and finally plated on marine agar supplemented with appropriate antibiotics. Incubated at 30˚C for 2 days.<br />
<br />
==Growth comparison==<br />
<br />
Growth comparison of ''Oceanibulbus indoliflex'' and ''Escherichia coli'' in marine and luria broth.<br />
We sought to compare the growth profiles of ''O. indoliflex'' and ''E. coli''. <br />
<br />
[[File:UniversityCollegeLondon_Marine_1.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_2.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_3.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_4.png|478px]]<br />
<br />
Figures 2-5. Comparative growth profiles of ''Escherichia coli K-12 substr. W3110'' and ''Oceanibulbus indoliflex'' in Marine Broth and LB media at 25°C and 37°C.<br />
<br />
==References==<br />
<br />
1. Brinkhoff, T., Giebel, H.-A., & Simon, M. (2008). Diversity, ecology, and genomics of the Roseobacter clade: a short overview. Archives of microbiology, 189(6), 531–9. <br />
<br />
2. Piekarski, T., Buchholz, I., Drepper, T., Schobert, M., Wagner-Doebler, I., Tielen, P., & Jahn, D. (2009). Genetic tools for the investigation of Roseobacter clade bacteria. BMC microbiology, 9, 265. <br />
<br />
3. Buchan, A., González, J. M., & Moran, M. A. (2005). Overview of the marine roseobacter lineage. Applied and environmental microbiology, 71(10), 5665–77. <br />
<br />
4. Dang, H., & Lovell, C. R. (2002). Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization. Environmental microbiology, 4(5), 287–95. <br />
<br />
5. Wagner-Döbler, I., & Biebl, H. (2006). Environmental biology of the marine Roseobacter lineage. Annual review of microbiology, 60, 255–80. <br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/AttributionsTeam:University College London/Attributions2012-09-27T03:50:36Z<p>Boo: </p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=portico}}<br />
<br />
All work has been conducted by the team except for contributions mentioned here. We would like to thank the following people and organisations for their assistance:<br />
<br />
Supervisors, Darren Nesbeth and Eli Keshavarz-Moore - requested funding from UCL Biochemical Engineering Department <br />
<br />
Yanika Borg, Erick Ramos and Alex Templar - our instructors <br />
<br />
Dr Paola Gomez-Perira, Researcher in Ocean Biogeochemistry and Ecosystems at Southampton University, advised Bouran on Marine Chassis and Bethan on Sediment Sampling and Experimental Design.<br />
<br />
Ben Mackrow, Research Associate in Algal Biotechnology at UCL, helped Bouran with Gibson Assembly and Construction feedback. <br />
<br />
Jason Gardiner and Douglas Ridgway, from the Alberta iGEM team, gave Bouran feedback on the Alberta BioBricks.<br />
<br />
Sean Ward and Markus Gershater, from Synthace, advised Bouran on Experimental Design and for help with electroporation. <br />
<br />
Prof. Mike Hoare, gave James and Leonard advice on creating a small scale shear device.<br />
<br />
Stuart Duffy, assisted in the creating and operation of the small scale shear device.<br />
<br />
<br />
== Modelling ==<br />
<br />
Dr David Rowley for help with the initial version of our degradation model<br />
<br />
Dr Andrew Martin for his assistance on running PERL scripts<br />
<br />
Miriam Goldstein for her help in finding data for the ocean model<br />
<br />
Stephen Fawcett for his help with ocean physics<br />
<br />
Julie Masura & Peter Ryan for their data on microplastic distribution<br />
<br />
Dr Chris Barnes for his tireless support with all aspects of our modelling<br />
<br />
<br />
== Human Practices ==<br />
<br />
Dr Muki Hacklay, advised Philipp, Bethan and Yeping on the development of our DIYbio - citizen science collaboration.<br />
<br />
Dr Steve Cross, <br />
<br />
Kate Oliver, from UCL Faculty of Engineering, advised Philipp and Bethan on publishing events to the public, finding resources within UCL and lent us tables for our Public BioBrick Exhibition. <br />
<br />
Kimberley Freeman and Hillary Jackson, from the UCL Department of Public Engagement, for their support and feedback for our Beacon Bursary application.<br />
<br />
Virgil Rerimassie & Dirk Stemerding, from the Rathenau Instituut in Den Haag, supported Erin with our application and development of our proposal for the Meeting of Young Minds debate, held by the Rathenau Institute. <br />
<br />
Claire Marris & Maggie Leggett for agreeing to provide support with our position paper for the Meeting of Young Minds debate<br />
<br />
Alan Evans at the National Oceanography Centre, Southampton, for his kind assistance on UNCLOS and international ocean law<br />
<br />
Mike Hughes, from Guerrilla Science, advised Yeping and Martina on public outreach and gave feedback on our DIYbio workshops.<br />
<br />
William Beaufoy, Simon Rose, Nicolas FitzRoy Dale, Andrew Cousins, Taylor Burcs and Joel Winston from the London Biohackers<br />
<br />
== Miscellaneous ==<br />
<br />
Prof Andrea Sella for providing us with dry ice and moral support<br />
<br />
Brian O'Sullivan & Gareth Mannall thanks to whom we now have a clean microwave<br />
<br />
Paula Thomas & Hayley Powell for their help getting rooms and equipment<br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boohttp://2012.igem.org/Team:University_College_London/Research/MarineBacteriaTeam:University College London/Research/MarineBacteria2012-09-27T03:24:04Z<p>Boo: /* Roseobacter clade bacteria */</p>
<hr />
<div>{{:Team:University_College_London/templates/head|coverpicture=training}}<br />
<br />
= Marine Chassis =<br />
<br />
==Roseobacter clade bacteria ==<br />
<br />
<br />
<br />
In consultation with Paola R. Gomez-Pereira of the National Oceanography Centre, Southampton, we identified ''Oceanibulbus indoliflex'' and ''Roseobacter dentitrificans'' as promising chassis for the expression of our systems. <br />
<br />
The roseobacter clade bacteria, RCB, constitute a significant proportion of coastal and ocean bacterioplankton communities, estimated above 20% and 15% respectively, and are found in a diverse range of marine habitats. (Buchan ''et al.'' 2006). RCB demonstrate numerous traits, including aerobic anoxygenic phototrophy, aromatic compound degradation and recycling of sulphur within the water column and have been implicated in carbon monoxide consumption. (Wagner-Döbler & Biebl, ''et al.'' 2006). Significantly, in the context of plastic Island, they are shown to be major colonisers of submerged surfaces in marine waters (Dang & Lovell ''et al.'' 2002).<br />
<br />
<br />
[[File:UniversityCollegeLondon_O_indolifex_Transformed_Wide.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Medium.jpg|310px]] [[File:UniversityCollegeLondon_O_indolifex_Transformed_Close.jpg|310px]]<br />
<br />
Figure 1. ''Oceanibulbus indoliflex''streaked on marine agar, showing colony morphology.<br />
<br />
==Transformation Protocols== <br />
<br />
Transformation of ''Oceanibulbus indoliflex'' by electroporation. Adapted from (Piekaski ''et al.'' 2009)<br />
<br />
Electrocompetent cells were prepared according to the following protocol: <br />
<br />
45ml Marine Broth was inoculated with 1ml of ''O. indoliflex'' and grown to ~ 0.5 at OD 578. <br />
<br />
10ml volumes were transferred to 50 mL falcons and cells were sedimented for 15mins at 3200 x g in a pre-chilled centrifuge. <br />
<br />
Cells were washed 5 times with 10% (v/v) glycerol, and finally resuspended in 400uL 10% (v/v) glycerol. <br />
<br />
50uL aliquots were made and stored at -80C. <br />
<br />
Electroporation:<br />
<br />
1uL DNA was added to 50uL competent cells to chilled 1mm electrocuvettes, and treated with a field strength of 2.5kV. <br />
<br />
1ml marine broth was added to cells and mixtures were transferred to falcons for incubation overnight at room temperature at 200rpm and finally plated on marine agar supplemented with appropriate antibiotics. Incubated at 30˚C for 2 days.<br />
<br />
==Growth comparison==<br />
<br />
Growth comparison of ''Oceanibulbus indoliflex'' and ''Escherichia coli'' in marine and luria broth.<br />
We sought to compare the growth profiles of ''O. indoliflex'' and ''E. coli''. <br />
<br />
[[File:UniversityCollegeLondon_Marine_1.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_2.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_3.png|478px]]<br />
[[File:UniversityCollegeLondon_Marine_4.png|478px]]<br />
<br />
Figures 2-5. Comparative growth profiles of ''Escherichia coli K-12 substr. W3110'' and ''Oceanibulbus indoliflex'' in Marine Broth and LB media at 25°C and 37°C.<br />
<br />
==References==<br />
<br />
1. Brinkhoff, T., Giebel, H.-A., & Simon, M. (2008). Diversity, ecology, and genomics of the Roseobacter clade: a short overview. Archives of microbiology, 189(6), 531–9. <br />
<br />
2. Piekarski, T., Buchholz, I., Drepper, T., Schobert, M., Wagner-Doebler, I., Tielen, P., & Jahn, D. (2009). Genetic tools for the investigation of Roseobacter clade bacteria. BMC microbiology, 9, 265. <br />
<br />
3. Buchan, A., González, J. M., & Moran, M. A. (2005). Overview of the marine roseobacter lineage. Applied and environmental microbiology, 71(10), 5665–77. <br />
<br />
4. Dang, H., & Lovell, C. R. (2002). Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization. Environmental microbiology, 4(5), 287–95. <br />
<br />
5. Wagner-Döbler, I., & Biebl, H. (2006). Environmental biology of the marine Roseobacter lineage. Annual review of microbiology, 60, 255–80. <br />
<br />
{{:Team:University_College_London/templates/foot}}</div>Boo