http://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&feed=atom&action=historyTeam:Bielefeld-Germany/Biosafety/Biosafety System - Revision history2024-03-29T06:33:37ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=361604&oldid=prevTore at 03:55, 29 October 20132013-10-29T03:55:00Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The induction of the Biosafety-Systems ''AraCtive'' and ''Lac of Growth'' with L-rhamnose leads to a repression of the expression of GFP when compared to the uninduced state. Therefore the second part of these Biosafety-Systems, which contains normally the toxic RNase Ba <bbpart>BBa_K1172904</bbpart> can be used as a kill-switch regulated by the pentose L-rhamnose. So it could be demonstrated that these Biosafety-Systems work in principle. Because of the tight repression of the second part by the arabinose promoter P<sub>''BAD''</sub> the Biosafety-System ''AraCtive'' would be probably the best choice at the moment. But there exists also powerful ideas for the others Biosafety-Systems like creating a [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Conclusions double ''lac'' promoter] to tighten the basal transcription and generating an Biosafety-System with a tight repression and a strong activation.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The induction of the Biosafety-Systems ''AraCtive'' and ''Lac of Growth'' with L-rhamnose leads to a repression of the expression of GFP when compared to the uninduced state. Therefore the second part of these Biosafety-Systems, which contains normally the toxic RNase Ba <bbpart>BBa_K1172904</bbpart> can be used as a kill-switch regulated by the pentose L-rhamnose. So it could be demonstrated that these Biosafety-Systems work in principle. Because of the tight repression of the second part by the arabinose promoter P<sub>''BAD''</sub> the Biosafety-System ''AraCtive'' <ins class="diffchange diffchange-inline"><bbpart>BBa-K1172909</bbpart> </ins>would be probably the best choice at the moment. But there exists also powerful ideas for the others Biosafety-Systems like creating a [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#Conclusions double ''lac'' promoter] to tighten the basal transcription and generating an Biosafety-System with a tight repression and a strong activation.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Another possibility could be the utilization of the Inhibitor Barstar instead of any repressor ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#References Mossakowska ''et. al'', 1989]). In this case are very tight repression of the Barstar is needed, when the cell leave the defined environment, to activate the Barnase. These approach probably open the possibility for a tight inhibition of the toxic gene product RNase Ba, but the activation of the Barnase might be very slow, because on the one hand the rhamnose promoter P<sub>''Rha''</sub> is continuously shut down, so that the inhibitor amount decreases only slowly and one the other hand the inhibitor Barstar is characterized by a slow degradation. A possible solution might be the introduction of a SsrA [http://parts.igem.org/Protein_domains/Degradation degradation tag] at the C-terminus of Alr ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#References Levchenko ''et. al'', 2003]).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Another possibility could be the utilization of the Inhibitor Barstar instead of any repressor ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#References Mossakowska ''et. al'', 1989]). In this case are very tight repression of the Barstar is needed, when the cell leave the defined environment, to activate the Barnase. These approach probably open the possibility for a tight inhibition of the toxic gene product RNase Ba, but the activation of the Barnase might be very slow, because on the one hand the rhamnose promoter P<sub>''Rha''</sub> is continuously shut down, so that the inhibitor amount decreases only slowly and one the other hand the inhibitor Barstar is characterized by a slow degradation. A possible solution might be the introduction of a SsrA [http://parts.igem.org/Protein_domains/Degradation degradation tag] at the C-terminus of Alr ([https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L#References Levchenko ''et. al'', 2003]).<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This approach might also useful for the degradation of the alanine racemase, because it can be seen, that all Biosafety-Systems can also grow without L-rhamnose induction, so that the double-kill switch mechanism is not ideal at this moment and a tighter repression of the alanine racemase is necessary to profit from the double-kill switch mechanism. As the high copy plasmid pSB1C3 was used for all Biosafety-Systems, it is [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions discussed] that an adjustment of the copy number might improve the double-kill switch mechanism. Moreover the lower plasmid copy number might simplify also the integration of the Barnase instead of GFP in the Biosafety-System.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This approach might also useful for the degradation of the alanine racemase, because it can be seen, that all Biosafety-Systems can also grow without L-rhamnose induction, so that the double-kill switch mechanism is not ideal at this moment and a tighter repression of the alanine racemase is necessary to profit from the double-kill switch mechanism. As the high copy plasmid pSB1C3 was used for all Biosafety-Systems, it is [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions discussed] that an adjustment of the copy number might improve the double-kill switch mechanism. Moreover the lower plasmid copy number might simplify also the integration of the Barnase instead of GFP in the Biosafety-System.<br></div></td></tr>
</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=361491&oldid=prevTore at 03:52, 29 October 20132013-10-29T03:52:07Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). In this case, on the one hand, the bacteria grow (F = 1) when the alanine racemase BBa_K1172901 is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other hand, the bacteria do not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) BBa_K1172904, or the absence of D-alanine..</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). In this case, on the one hand, the bacteria grow (F = 1) when the alanine racemase <ins class="diffchange diffchange-inline"><bbpart></ins>BBa_K1172901<ins class="diffchange diffchange-inline"></bbpart> </ins>is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other hand, the bacteria do not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <ins class="diffchange diffchange-inline"><bbpart></ins>BBa_K1172904<ins class="diffchange diffchange-inline"></bbpart></ins>, or the absence of D-alanine..</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td></tr>
</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360993&oldid=prevTore at 03:39, 29 October 20132013-10-29T03:39:38Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). <del class="diffchange diffchange-inline">This shows that </del>the bacteria grow (F = 1) when the alanine racemase <del class="diffchange diffchange-inline"><bbpart></del>BBa_K1172901<del class="diffchange diffchange-inline"></bbpart> </del>is expressed (C = 1) or D-alanine is supplemented (D<del class="diffchange diffchange-inline">&nbsp;</del>=<del class="diffchange diffchange-inline">&nbsp;</del>1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other <del class="diffchange diffchange-inline">side </del>the bacteria <del class="diffchange diffchange-inline">does </del>not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <del class="diffchange diffchange-inline"><bbpart></del>BBa_K1172904<del class="diffchange diffchange-inline"></bbpart></del>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). <ins class="diffchange diffchange-inline">In this case, on the one hand, </ins>the bacteria grow (F = 1) when the alanine racemase BBa_K1172901 is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other <ins class="diffchange diffchange-inline">hand, </ins>the bacteria <ins class="diffchange diffchange-inline">do </ins>not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) BBa_K1172904, or the absence of D-alanine<ins class="diffchange diffchange-inline">.</ins>.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td></tr>
</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360795&oldid=prevTore at 03:33, 29 October 20132013-10-29T03:33:54Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D =&nbsp;1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D<ins class="diffchange diffchange-inline">&nbsp;</ins>=&nbsp;1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td></tr>
</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360766&oldid=prevTore at 03:33, 29 October 20132013-10-29T03:33:06Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D =<ins class="diffchange diffchange-inline">&nbsp;</ins>1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth.]]</div></td></tr>
</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360715&oldid=prevTore at 03:31, 29 October 20132013-10-29T03:31:37Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|600px|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism<ins class="diffchange diffchange-inline">. Inputs are represented by the expression of the Barnase (A) and alanine racemase (C), while the output represents the bacterial growth</ins>.]]</div></td></tr>
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</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360589&oldid=prevTore at 03:27, 29 October 20132013-10-29T03:27:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png<ins class="diffchange diffchange-inline">|600px</ins>|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism.]]</div></td></tr>
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</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360560&oldid=prevTore at 03:27, 29 October 20132013-10-29T03:27:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (A = 1) or the alanine racemase repressed (C = 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (C = 1) or D-alanine is supplemented (D = 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (B = 1), like L-arabinose, the expression of the RNase Ba (A = 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[File:Team-Bielefeld-Biosafety-Systems-logical-gates.png|thumb|center|'''Figure 4:''' Logical gate of the double kill-switch mechanism.]]</ins></div></td></tr>
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</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360246&oldid=prevTore at 03:19, 29 October 20132013-10-29T03:19:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (<del class="diffchange diffchange-inline">x </del>= 1) or the alanine racemase repressed (<del class="diffchange diffchange-inline">x </del>= 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (<del class="diffchange diffchange-inline">x </del>= 1) or D-alanine is supplemented (<del class="diffchange diffchange-inline">x </del>= 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (<del class="diffchange diffchange-inline">x </del>= 1), like L-arabinose, the expression of the RNase Ba (<del class="diffchange diffchange-inline">x </del>= 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (E = 1) and do not when the RNase Ba is expressed (<ins class="diffchange diffchange-inline">A </ins>= 1) or the alanine racemase repressed (<ins class="diffchange diffchange-inline">C </ins>= 0). This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow (F = 1) when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed (<ins class="diffchange diffchange-inline">C </ins>= 1) or D-alanine is supplemented (<ins class="diffchange diffchange-inline">D </ins>= 1) to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow (F = 0), when induced by an inducer (<ins class="diffchange diffchange-inline">B </ins>= 1), like L-arabinose, the expression of the RNase Ba (<ins class="diffchange diffchange-inline">A </ins>= 1) <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following the results of the three Biosafety-Systems [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S ''AraCtive''], [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_M ''TetOR alive''] and [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L ''Lac of Growth''] a compared together to get an impression of their difference. Therefore, the metabolic pressure of the Biosafety-Plasmids, their basal transcription and the kill-switch mechanism were simulated with GFP <bbpart>BBa_E0040</bbpart>.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Following the results of the three Biosafety-Systems [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S ''AraCtive''], [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_M ''TetOR alive''] and [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L ''Lac of Growth''] a compared together to get an impression of their difference. Therefore, the metabolic pressure of the Biosafety-Plasmids, their basal transcription and the kill-switch mechanism were simulated with GFP <bbpart>BBa_E0040</bbpart>.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">4 </del>compares the cultivation process of the Biosafety-Strain ''E. coli'' K-12 ∆''alr'' ∆''dadX'' containing the corresponding Biosafety-Plasmid. The cells were cultivated in M9 minimal medium with glycerol and 1% L-rhamnose to measure the metabolic pressure caused by the Biosafety-Plasmid. In the presence of L-rhamnose, the expression of the repressor and the alanine racemase (Alr) is induced, while the expression of the toxic gene product is repressed. Compared to the uninduced Biosafety-Systems the expression of these genes causes a higher energy expenditure for the cell, but as demonstrated in Figure 6 there is no significant difference between the Biosafety-Systems. So metabolic pressure is not a criteria for the preference of one of the Biosafety-Systems.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">5 </ins>compares the cultivation process of the Biosafety-Strain ''E. coli'' K-12 ∆''alr'' ∆''dadX'' containing the corresponding Biosafety-Plasmid. The cells were cultivated in M9 minimal medium with glycerol and 1% L-rhamnose to measure the metabolic pressure caused by the Biosafety-Plasmid. In the presence of L-rhamnose, the expression of the repressor and the alanine racemase (Alr) is induced, while the expression of the toxic gene product is repressed. Compared to the uninduced Biosafety-Systems the expression of these genes causes a higher energy expenditure for the cell, but as demonstrated in Figure 6 there is no significant difference between the Biosafety-Systems. So metabolic pressure is not a criteria for the preference of one of the Biosafety-Systems.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafeyt-System-WachstumALL.jpg|600px|thumb|center|'''Figure <del class="diffchange diffchange-inline">6</del>:''' Comparision of the metabolic pressure caused by the corresponding Biosafety-System. Shown are the cultivation in M9 minimal medium with an induction of 1% L-rhamnose, so that the first part of the System is expressed and the second repressed. All Biosafety-Systems show about the same growth.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafeyt-System-WachstumALL.jpg|600px|thumb|center|'''Figure <ins class="diffchange diffchange-inline">5</ins>:''' Comparision of the metabolic pressure caused by the corresponding Biosafety-System. Shown are the cultivation in M9 minimal medium with an induction of 1% L-rhamnose, so that the first part of the System is expressed and the second repressed. All Biosafety-Systems show about the same growth.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p align="justify"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In Figure <del class="diffchange diffchange-inline">7</del>, the basal transcription of the three Biosafety-Systems is analyzed by comparison of the specific production rate of GFP. This is important to get an impression of the lethality of each Biosafety-System, because a higher basal transcription will result in a higher expression of the toxic RNase Ba <bbpart>BBa_K1172904</bbpart>. Therefore, this parameter is even more important for the function of the Biosafety-System. Figure <del class="diffchange diffchange-inline">5 </del>demonstrated that the Biosafety-System ''AraCtive'' is characterized by the lowest basal transcription, followed by the Biosafety-System ''Lac of Growth'' and ''TetOR alive''. This means that the Biosafety-System ''AraCtive'' <bbpart>BBa_K1172909</bbpart> would be the most potent one, as the toxic gene product will have the lowest effect and therefore the growth rate will be the highest in this System.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In Figure <ins class="diffchange diffchange-inline">6</ins>, the basal transcription of the three Biosafety-Systems is analyzed by comparison of the specific production rate of GFP. This is important to get an impression of the lethality of each Biosafety-System, because a higher basal transcription will result in a higher expression of the toxic RNase Ba <bbpart>BBa_K1172904</bbpart>. Therefore, this parameter is even more important for the function of the Biosafety-System. Figure <ins class="diffchange diffchange-inline">6 </ins>demonstrated that the Biosafety-System ''AraCtive'' is characterized by the lowest basal transcription, followed by the Biosafety-System ''Lac of Growth'' and ''TetOR alive''. This means that the Biosafety-System ''AraCtive'' <bbpart>BBa_K1172909</bbpart> would be the most potent one, as the toxic gene product will have the lowest effect and therefore the growth rate will be the highest in this System.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-sezProduktbildungALL.jpg|600px|thumb|center|'''Figure <del class="diffchange diffchange-inline">7</del>:''' Comparision of the specific production rate of the three Biosafety-Systems. Shown are the cultivation in M9 minimal medium with an induction of 1% L-rhamnose. Obviously the Biosafety-System ''AraCtive'' is characterized by the lowest basal transcription followed by the Biosafety-Systemas ''Lac of Growth'' and ''TetOR alive''.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafety-Systems-sezProduktbildungALL.jpg|600px|thumb|center|'''Figure <ins class="diffchange diffchange-inline">6</ins>:''' Comparision of the specific production rate of the three Biosafety-Systems. Shown are the cultivation in M9 minimal medium with an induction of 1% L-rhamnose. Obviously the Biosafety-System ''AraCtive'' is characterized by the lowest basal transcription followed by the Biosafety-Systemas ''Lac of Growth'' and ''TetOR alive''.]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Moreover, the kill-switch mechanism is another important characteristic of the Biosafety-Systems. This parameter can be measured by difference in the expression levels of GFP. An ideal Biosafety-System would show a minimal basal transcription in the presence of L-rhamnose and a high expression level in its absence to ensure the kill mechanism. The kill-switch mechanisms for the three Biosafety-Systems are shown in Figure <del class="diffchange diffchange-inline">8</del>. All Biosafety-Systems were cultivated in M9 minimal medium. The bars marked with a star are additional induced with 1% L-rhamnose, resulting in the expression of the first part, the repressor and the alanine racemase. It can be seen that the Biosafety-System ''AraCtive'' with the lowest basal transcription (orange bar) shows a higher expression in the absence of L-rhamnose (red bar), but the activation is not as strong compared to Biosafety-System ''Lac of Growth''. This Biosafety-System is characterized by a higher basal transcription in the presence of L-rhamnose (light blue bar), but a potent activation in its absence (dark blue bar). The Biosafety-System ''TetOR alive'' shows the highest basal transcription and the activation differs not that much during cultivation. Because the TetOR-System is actually known for an effective switch mechanism, the optimizations of the Biosafety-System are [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_M discussed] and needs optimizations before an adequate analysis is possible.</p><br> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Moreover, the kill-switch mechanism is another important characteristic of the Biosafety-Systems. This parameter can be measured by difference in the expression levels of GFP. An ideal Biosafety-System would show a minimal basal transcription in the presence of L-rhamnose and a high expression level in its absence to ensure the kill mechanism. The kill-switch mechanisms for the three Biosafety-Systems are shown in Figure <ins class="diffchange diffchange-inline">7</ins>. All Biosafety-Systems were cultivated in M9 minimal medium. The bars marked with a star are additional induced with 1% L-rhamnose, resulting in the expression of the first part, the repressor and the alanine racemase. It can be seen that the Biosafety-System ''AraCtive'' with the lowest basal transcription (orange bar) shows a higher expression in the absence of L-rhamnose (red bar), but the activation is not as strong compared to Biosafety-System ''Lac of Growth''. This Biosafety-System is characterized by a higher basal transcription in the presence of L-rhamnose (light blue bar), but a potent activation in its absence (dark blue bar). The Biosafety-System ''TetOR alive'' shows the highest basal transcription and the activation differs not that much during cultivation. Because the TetOR-System is actually known for an effective switch mechanism, the optimizations of the Biosafety-System are [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_M discussed] and needs optimizations before an adequate analysis is possible.</p><br> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafeyt-System-Gesamt-Balken.jpg|600px|thumb|center|'''Figure <del class="diffchange diffchange-inline">8</del>:''' Comparison of all three Biosafety-Systems. The Figure shows the specific production rate of the induced (1% L-rhamnose) Biosafety-System (marked with a star) and the uninduced Biosafety-Systems.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:Team-Bielefeld-Biosafeyt-System-Gesamt-Balken.jpg|600px|thumb|center|'''Figure <ins class="diffchange diffchange-inline">7</ins>:''' Comparison of all three Biosafety-Systems. The Figure shows the specific production rate of the induced (1% L-rhamnose) Biosafety-System (marked with a star) and the uninduced Biosafety-Systems.]]</div></td></tr>
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</table>Torehttp://2013.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Biosafety/Biosafety_System&diff=360001&oldid=prevTore at 03:05, 29 October 20132013-10-29T03:05:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Logical gate===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (1) and do not when the RNase Ba is expressed or the alanine racemase repressed. This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed or D-alanine is supplemented to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow, when induced by an inducer, like L-arabinose, the expression of the RNase Ba <bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Besides the genetical approach the constructed Biosafety-System can also function as a logical gate, as shown in Figure 4. So only when the alanine racemase (Alr) is expressed the bacteria grow (<ins class="diffchange diffchange-inline">E = </ins>1) and do not when the RNase Ba is expressed <ins class="diffchange diffchange-inline">(x = 1) </ins>or the alanine racemase repressed <ins class="diffchange diffchange-inline">(x = 0)</ins>. This concept can be extended with the addition of substances like D-alanine or an inducer, like L-arabinose for the Biosafety-System AraCtive (Figure 5). This shows that the bacteria grow <ins class="diffchange diffchange-inline">(F = 1) </ins>when the alanine racemase <bbpart>BBa_K1172901</bbpart> is expressed <ins class="diffchange diffchange-inline">(x = 1) </ins>or D-alanine is supplemented <ins class="diffchange diffchange-inline">(x = 1) </ins>to the medium, allowing bacterial growth also in the absence of L-rhamnose. On the other side the bacteria does not grow <ins class="diffchange diffchange-inline">(F = 0)</ins>, when induced by an inducer <ins class="diffchange diffchange-inline">(x = 1)</ins>, like L-arabinose, the expression of the RNase Ba <ins class="diffchange diffchange-inline">(x = 1) </ins><bbpart>BBa_K1172904</bbpart>, or the absence of D-alanine.</p><br></div></td></tr>
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