Difference between revisions of "Part:BBa K1172909"
 
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__NOTOC__
 
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<partinfo>BBa_K1172909 short</partinfo>
 
<partinfo>BBa_K1172909 short</partinfo>
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<p align="justify">
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The Biosafety-System araCtive <bbpart>BBa_K1172909</bbpart> is an improvement of the BioBrick <bbpart>BBa_K914014</bbpart> by replacing:
  
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These biobrick is an improvement of the Biobrick <bbpart>BBa_K914014</bbpart> by changing:
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*the first promoter arabinose P<sub>''BAD''</sub> by the rhamnose promoter P<sub>''Rha''</sub> to obtain a lower basal transcription.
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*the promoter to obtain a lower basal transcription.
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* intgration of the alanine racemase <bbpart>BBa_K1172901</bbpart>(''alr'') for gaining higher plasmid stability and for taking advantage of the double-kill switch.
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*the Alanine-Racemase <bbpart>BBa_K1172901</bbpart>(''alr'') for gaining higher plasmid stability and for taking advantage of the double-kill switch.
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*the repressor LacI to AraC and the ''lac'' promoter to the arabinose promoter P<sub>''BAD''</sub> for a higher repression and lower basal transcription of the second part.
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*changing the repressor lacI to araC and the lac promoter to the arabinose pBAD promoer for higher repression and lower basal transcription of the second part.
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<br>
 
<br>
 +
For more details about the seperate genes in this Biosafety-System and their function, click [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S here]
 +
<br><br>
 +
For more details about the function of the Biosafety-System in general, click [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System here]</p><br>
  
  
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<span class='h3bb'>'''Sequence and Features'''</span>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1172909 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1172909 SequenceAndFeatures</partinfo>
  
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<br>
 
<br>
 
<p align="justify">
 
<p align="justify">
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Together with the Biosafety-Strain K-12 ∆alr ∆dadX the Biosafety-System araCtive takes advantage of this genes by combine them to a powerful device, which allows to control the bacterial cell division. The control of the bacterial growth is thereby active and passive possible. Active by inducing the pBAD promoter with L-arabinose and passive by the induction of L-Rhamnose. The passive control makes it possible to control the bacterial cell division in an defined environment, like the MFC by adding continously L-Rhamnose to the media. As shown in the figure below, this leads to an expression of the essential Alanine-Racemase (''alr'') and the araC repressor, so that the expression of the RNase Ba is repressed. </p><br>
+
Combining the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S genes] described above with the Biosafety-Strain K-12 ∆''alr'' ∆''dadX'' results in a powerful device, allowing us to control the bacterial cell division. The control of the bacterial growth is possible either active or passive. Active by inducing the P<sub>''BAD''</sub> promoter with L-arabinose and passive by the induction of L-rhamnose. The passive control makes it possible to control the bacterial cell division in a defined closed environment, like the MFC, by continuously adding L-rhamnose to the medium. As shown in the Figure 1 below, this leads to an expression of the essential alanine racemase (''alr'') and the AraC regulator, so that the expression of the RNase Ba is repressed. </p><br>
  
−
[[File:IGEM Bielefeld 2013 Biosafety System S+ 2.png|600px|thumb|center|'''Figure 10:''' Biosafety-System araCtive in the presens of L-Rhamnose. The essentail Alanine-Racemase (''alr'') and the repressor araC are expressed, so that the expression of the RNAse Ba is repressed and the Bacteria grow.]]
+
 
 +
[[File:IGEM Bielefeld 2013 Biosafety System S+ 2.png|600px|thumb|center|'''Figure 1:''' Biosafety-System araCtive in the presence of L-rhamnose. The essential alanine racemase (Alr) and the repressor AraC are expressed, resulting in a repression of the expression of the RNAse Ba. Consequently the bacteria show normal growth behaviour.]]
  
 
<br>
 
<br>
  
 
<p align="justify">
 
<p align="justify">
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When the bacteria exit the defined environment of the MFC or L-Rhamnose is not added any more to the media, the expression of the Alanine-Racemase (''alr'') and the araC repressor decreases, so that the expression of the toxic RNase Ba (Barnase) is not inhibted so strong any more. The cleavage of the intracellular RNA by the Barnase and ideally also the lack of synthesized D-alanine, caused by the repressed Alanine-Racemase inhibits the cell division and makes sure that the bacteria can only grow in the defined area. </p><br>
+
In the event that bacteria exit the defined environment of the MFC or L-rhamnose is not added to the medium any more, both the expression of the alanine racemase (Alr) and the AraC regulatordecrease, so that the expression of the toxic RNase Ba (Barnase) begins. The cleavage of the intracellular RNA by the Barnase and the lack of synthesized D-alanine, caused by the repressed alanine racemase inhibit the cell division and makes sure that the bacteria can only grow in the defined environment or the device of choice respectively. </p><br>
  
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[[File:IGEM Bielefeld 2013 Biosafety System S ohne Rhamnose 2.png|600px|thumb|center|'''Figure 11:''' Biosafety-System araCtive outside the defined conditions and a decreased concentration of L-Rhamnose. The expression of the Alanine-Racemase and araC repressor is reduced and ideally completly shutdown. In contrast the expression of the RNase ba (Barnase is sligthly turned on, leading to cell death by RNA cleavage.]]
+
[[File:IGEM Bielefeld 2013 Biosafety System S ohne Rhamnose 2.png|600px|thumb|center|'''Figure 2:''' Active Biosafety-System araCtive outside of a defined environment lacking L-rhamnose. Both the expression of the alanine racemase (Alr) and AraC repressor are reduced and ideally completely shutdown. In contrast, the expression of the RNase ba (Barnase) is turned on, leading to cell death by RNA cleavage.]]
  
 
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==Results==
 
==Results==
 
<br>
 
<br>
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==='''Characterization of the arabinose promoter pBAD'''===
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==='''Characterization of the arabinose promoter P<sub>''BAD''</sub>'''===
  
 
<p align="justify">
 
<p align="justify">
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First of all the bacterial growth under the pressure of the unrepressed pBAD promoter was investigated on different carbon source. Therefore the cultivation on M9 minimal media with Glucose or Glycerol was characterized. To identify the transcription rate of the unrepressed arabionse promoter pBAD the expression of the green fluorescence protein GFP <bbpart>BBa_E0040</bbpart> behind the pBAD promoter was used.<br>
+
First of all, the arabinose promoter P<sub>''BAD''</sub> was characterized to get a first impression of its basal transcription rate. Therefore the bacterial growth was investigated under the pressure of the unrepressed P<sub>''BAD''</sub> promoter on different carbon source using M9 minimal medium with either glucose or glycerol. The transcription rate was determined by fluorescence measurement of GFP <bbpart>BBa_E0040</bbpart> transcribed via the P<sub>''BAD''</sub> promoter using the BioBrick <bbpart>BBa_I13541</bbpart>.<br>
−
As shown in figure 12 below, the bacteria adapted better on glucose then on Glycerol. As glucose is the more powerful energy source, because it posses more carbon atoms than glycerol these result was expected before. So more interesting are the fluorescence measurement shown in the figure 13. As it can be seen both the wild type K-12 as the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC show about the same fluorescence on glucose (blue and black curve) but differ on glycerin. This can be explained by the fact that glucose itself also represses the arabinose promoter, while glycerol does not. In the presence of glucose the intracellular concentration of cAMP is low to repress the inefficient catabolism of arabinose, so that the glucose is catabolized first by the bacteria resulting in an optimal growth. In the absence of glucose increases, which enhances the transcription of the most operons, who regulate the enzymes for the catabolism of an alternative carbon source. Therefore the expression of GFP under the control of the pBAD promoter decreases on glycerol. Another different can be seen between the wild type (orange curve) and the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC (red curve). The Biosafety-Strain shows lower expression then the wild type, but has according to figure 12 about the same growth rate. This is caused by the fact that the araC gene in the Biosafety-Strain was deleted an as the araC gene functions not only as are repressor but also as an activator the transcription rate decreases.</p><br>
+
 
 +
As shown in Figure 3 below, the bacteria grew better on glucose than on glycerol. This is caused by the fact that glucose is the better energy source of this two, because glycerol enters glycolysis at a later step and therefore delivers less energy (in addition to the additional ATP consumption to drive glycerol uptake). For the investigation of the basal transcription the fluorescence measurements, shown in Figure 4, is more interesting. <br>
 +
Both the wild type ''E. coli'' K-12 and the strain ''E. coli'' K-12 ∆''alr'' ∆''dadX'' ∆''araC'' show about the same fluorescence on glucose (red and orange curve) but differ on glycerol. This can be explained by the fact that growth on glucose does not cause an additional induction of the arabinose promoter P<sub>''BAD''</sub>, while glycerol does. In the presence of glucose, the intracellular concentration of cAMP is low. The absence of cAMP results in inactive CAP (carbon utilization activator protein) and thus no induction of inefficient catabolisms like the catabolism of arabinose. Therefore, glucose is catabolized by the bacteria first, resulting in an optimal growth. In the absence of glucose, the cAMP level increases which enhances via CAP-cAMP the transcription of most operons encoding enzymes for the catabolism of alternative carbon sources. Therefore, the expression of GFP under the control of the P<sub>BAD</sub> promoter increases on glycerol. Another difference can be seen between the wild type (black curve) and the strain K-12 ∆''alr'' ∆''dadX'' ∆''araC'' (blue curve).  
 +
The Biosafety-Strain shows lower expression then the wild type, but has about the same growth rate, according to Figure 11. The reason for this characteristic is caused by the deleted AraC protein in the Biosafety-Strain. Because the AraC protein functions not only as are repressor but also as an activator the transcription rate decreases in the Biosafety-Strain.</p><br>
 +
 
  
 
<br>
 
<br>
  
−
[[File:Team-Bielefeld-Biosafety-System_araCtive_OD.jpg|600px|thumb|center|'''Figure 12:''' Characterization of the bacterial growth of the pBAD promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC. The M9 media was supplemented with 5 mM D-alanine. It can be seen, that the bacteria grow faster on M9 minimal media glucose than on M9 minimal media glycerol.]]
+
[[File:Team-Bielefeld-Biosafety-System_araCtive_OD.jpg|600px|thumb|center|'''Figure 3:''' Characterization of the bacterial growth of the strain K-12 ∆''alr'' ∆''dadX'' ∆''araC'' containing the plasmid <bbpart>BBa_I13541</bbpart> with GFP (<bbpart>BBa_E0040</bbpart>) under the control of the P<sub>''BAD''</sub> promoter. The M9 medium was supplemented with 5 mM D-alanine. It could be demonstrated, that the bacteria grow faster on M9 minimal medium containing glucose than on M9 minimal medium with glycerol.]]
 
<br>
 
<br>
−
[[File:Team-Bielefeld_Biosafety-System-araCtive_fluorescence.jpg|600px|thumb|center|'''Figure 13:''' Characterization of the fluorescence of pBAD promoter with GFP (<bbpart>BBa_E0040</bbpart>) in the Biosafety-Strain K-12 ∆alr ∆dadX ∆araC. The bacteria were cultivated on M9 minimal media with 5 mM D-alanine supplemented.]]
+
[[File:Team-Bielefeld_Biosafety-System-araCtive_fluorescence.jpg|600px|thumb|center|'''Figure 4:''' Characterization of the fluorescence of the strain K-12 ∆''alr'' ∆''dadX'' ∆''araC'' containing the plasmid <bbpart>BBa_I13541</bbpart> with GFP (<bbpart>BBa_E0040</bbpart>) under control of the P<sub>''BAD''</sub> promoter. The Biosafety-Strain was cultivated on M9 minimal medium with 5 mM D-alanine supplemented.]]
 
<br>
 
<br>
  
 
<p align="justify">
 
<p align="justify">
−
The effect that glucose represses the transcription of the pBAD promoter becomes more clear by the specific production rate, as shown in figure 14. The specific production rate was thereby calculated via equation (1) :<br>
+
The effect that glucose represses the transcription of the P<sub>''BAD''</sub> promoter becomes more obvious by calculating of the specific production rates of GFP, as shown in Figure 5. The specific production rates were calculated via equation (1) :</p><br>
  
−
[[File:IGEM Bielefeld 2013 Sepzifische Produktionsrate.png|600px|center|]]
+
[[File:IGEM Bielefeld 2013 Sepzifische Produktionsrat.png|600px|center|]]
 
<br>
 
<br>
 +
<p align="justify">
 +
With the specific production rate of GFP, it can be demonstrated that GFP synthesis rates differ extremely between the cultivation on glucose and glycerol. The specific production rate is constantly very low when using glucose as carbon source, but shows a higher more unstable level in the cultivation with glycerol.<br>
 +
Because the specific production rate was calculated between every single measurement point, the curve in Figure 5 is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. And as neither of this curves are ideal, the fluctuations are the result. Nevertheless this graph shows clearly the difference between the two carbon sources.</p><br>
  
−
 
+
[[File:Team-Bielefeld-Biosafety-System-araCtive-pBAD-GFProduct.jpg|600px|thumb|center|'''Figure 5:''' Specific production rate of GFP transcribed via the P<sub>BAD</sub> promoter in dependence of different carbon sources.]]
−
So it can be seen that the building of GFP differs extremly between the cultivation on glucose and the cultivation of glycerol. While the production rate by using glucose as carbon source is nearly constant very low, it is thereby high in the cultivation with glycerol. As the specific production rate was calculated between every single measurement point the curve is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the transcription in the expression of GFP. They are caused by the growth curve and the fluorescence curve. And as they are not ideal there exists the fluctuations. But this graph shows clearly the difference between the two carbon sources.</p><br>
+
−
[[File:Team-Bielefeld-Biosafety-System-araCtive-pBAD-GFProduct.jpg|600px|thumb|center|'''Figure 14:''' Specific growth rate of GFP behind the pBAD promoter by the use of different carbon sources.]]
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<br>
 
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<br>
 
<br>
 
<p align="justify">
 
<p align="justify">
−
The Biosafety-System araCtive was characterized on M9 minimal medium with glycerol as carbon source. As for the characterization of the pure arabinose promoter pBAD above, the bacterial growth and the fluorescence of GFP <bbpart>BBa-E0040</bbpart> was measured. Therefore the wild type and the Biosafety-Strain ''E. coli'' K-12 ∆alr ∆dadX were cultivated once with the induction of 1% L-Rhamnose and once only on glycerol.
+
The Biosafety-System araCtive was characterized on M9 minimal medium using glycerol as carbon source. As for the characterization of the pure arabinose promoter P<sub>''BAD''</sub> above, the bacterial growth and the fluorescence of GFP <bbpart>BBa_E0040</bbpart> was measured. Therefore, the wild type and the Biosafety-Strain ''E. coli'' K-12 ∆''alr'' ∆''dadX'', both containing the Biosafety-Plasmid <bbpart>BBa_K1172909</bbpart>, were cultivated once with the induction of 1% L-rhamnose and once only on glycerol.<br>
−
First of all it is obviously shown in figure 15 that the growth of the bacteria, who are induced with 1 % L-Rhamnose (blue and black curve) is significant slower than on pure glycerol (orange and red curve). This is attributed to the high metabolic pressure of the induced bacteria. The expression of the repressor araC and the Alanine-Racemase (''alr'') simultaneously causes a high outlay of the cells so that they grow slower then the uninduced cells, who expresses only GFP. Additional the arabinose promoter pBAD is tightly regulated, so that the expression even with a small amount of the repressor AraC is not that high and therefore not as stressful. <br>
+
It becomes obvious (Figure 6) that the bacteria, induced with 1 % L-rhamnose (red and black curve) grow significantly slower than on pure glycerol (orange and blue curve). This is attributed to the high metabolic burden encountered by the induced bacteria. The expression of the repressor AraC and the alanine racemase (Alr) simultaneously causes a high strain on the cells, so that they grow slower than the uninduced cells, which express only GFP.<br>
−
Comparing the bacterial growth with the fluorescence in figure 16 it can be seen that the fluorescence seems to follow the same figure than the bacterial growth. The uninduced cells shows approximately an exponantial rise of fluorescence, while the fluorescence of the induced bacteria increases only slow.</p><br>
+
Comparing the bacterial growth with the fluorescence in Figure 7, it can be seen that the fluorescence seems to follow the same trend than the bacterial growth. The uninduced cells show approximately an exponential rise of fluorescence, while in comparision the fluorescence of the induced bacteria increases only slowly.</p><br>
  
−
[[File:Team-Bielefeld-Biosafety-System-araCtive-ODALL.jpg|600px|thumb|center|'''Figure 15:''' Characterization of the bacterial growth of the Biosafety-System on M9 minimal media glycerol. The figure compares the wild tpye k-12 and the Biosafety-Strain K-12 ∆alr ∆dadX and the induction by L-Rhamnose to pure glycerol.]]
+
[[File:Team-Bielefeld-Biosafety-System-araCtive-ODALL.jpg|600px|thumb|center|'''Figure 6:''' Characterization of the bacterial growth of the Biosafety-System on M9 minimal medium with glycerol. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆''alr'' ∆''dadX'' containing the Biosaftey-Plasmid <bbpart>BBa_K1172909</bbpart> and the induction by 1% L-rhamnose to pure glycerol.]]
 
<br>
 
<br>
−
[[File:Team-Bielefeld-Biosafety-System-araCtive-FlourescenceALL.jpg|600px|thumb|center|'''Figure 16:''' Characterization of the fluorescence of the Biosafety-System araCtive. The figure compares the wild tpye k-12 and the Biosafety-Strain K-12 ∆alr ∆dadX and the induction by L-Rhamnose to pure glycerol.]]
+
[[File:Team-Bielefeld-Biosafety-System-araCtive-FlourescenceALL.jpg|600px|thumb|center|'''Figure 7:''' Characterization of the fluorescence of the Biosafety-System araCtive. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆''alr'' ∆''dadX'' containing the Biosafety-Plasmid <bbpart>BBa_K1172909</bbpart> and the induction by 1% L-rhamnose to pure glycerol..]]
 
<br>
 
<br>
 
<p align="justify">
 
<p align="justify">
−
From the figure above it can not be seen if the expression of the repressor araC does effect the transcription of GFP or not. The slower growth of the bacteria is a first indication that the repressor araC and the Alanine-Racemase are highly expressed, but as the growth of the bacteria shows nearly the same figure than the fluorescence it could be possible that the repressor does not effect the expression level of GFP under the control of the arabinose promoter pBAD. That the Biosafety-System works as aspected by repressing the expression of GFP in the presence of L-Rhamnose can be seen from figure 18 below. The calculated specific production rate (equation 1) differs clearly, so that the production of GFP in the presence of L-Rhamnose is always lower than in its absence. <br>
+
From the data presented above it can not be determined if the expression of the repressor AraC does affect the transcription of GFP or not. The slower growth of the bacteria is a first indication that the repressor AraC and the alanine racemase (Alr) are highly expressed, but the growth of the bacteria shows nearly the same kinetics as the fluorescence. So it could be possible that the repressor does not affect the expression level of GFP under the control of the arabinose promoter P<sub>''BAD''</sub>. This becomes more clear by the calculation of the specific production rate of GFP by equation (1) . As shown in Figure 8 below the specific production rate differs clearly between the uninduced Biosafety-System and the Biosafety-System induced by 1% L-rhamnose. The production of GFP in the presence of L-rhamnose (red curve) is always lower than in its absence (orange curve), so that the expression of GFP is repressed in the presence of L-rhamnose.<br>
  
−
As the specific production rate was calculated between every single measurement point the curve is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the transcription in the expression of GFP. They are caused by the growth curve and the fluorescence curve. And this measured curves are not ideal the calculation of the specific production rate causes the fluctuations. But it can be seen very clear that the production of GFP differ an is much lower, when the bacteria are induced with 1% L-Rhamnose. So the Biosafety-System araCtive works.</p><br>
+
Because the specific production rate of GFP was calculated between every single measurement point, the curve in Figure 8 is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. But there is a clear tendency that the production of GFP is significantly lower when the bacteria are induced with 1% L-rhamnose. So the Biosafety-System araCtive works.</p><br>
  
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[[File:Team-Bielefeld-Biosafety-System-araCtive-spezProductSara.jpg|600px|thumb|center|'''Figure 17:''' Specific production rate, calculated by equation (1). The production rate of GFP of the uninduced bacteria is significant higher compared to the bacteria induced with 1% L-Rhamnose. The Biosafety-System AraCtive works.]]
+
[[File:Team-Bielefeld-Biosafety-System-araCtive-spezProductSara.jpg|600px|thumb|center|'''Figure 8:''' Specific production rate of GFP for the Biosafety-System araCtive, calculated via equation (1). The production rate of GFP of the uninduced bacteria is significantly higher compared to the bacteria induced with 1% L-rhamnose. The Biosafety-System AraCtive works.]]
  
 
==='''Conclusion of the Results'''===
 
==='''Conclusion of the Results'''===
−
<br>
 
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<p align="justify">
 
−
As the expression level of GFP is increased in the absence of L-Rhamnose and decreased in its presence, the Biosafety-System araCtive works as aspected. In figure 18 the specific production rates after 7,5 hours are compared.  It can be seen that the expression level of the pBAD promoter decreases in the uninduced Safety-Strain compared to the uninduced second part of the Biosafety-System and that the induction with L-Rhamnose leads to a tight repression of the transcription and therefore the expression of GFP.</p><br>
 
  
−
[[File:Team-Bielefeld-Biosafety-System-araCtive-Resultbalken.jpg|600px|thumb|center|'''Figure 18:''' Comparision of the specific production rate of GFP in the with L-Rhamnose induced Biosafety-System araCtive, the uninduced Biosafety-System araCtive and the second part of the Biosafety-System (pBAD-GFP only).]]
+
For the conclusion of the result, visit our [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions wiki].
  
  
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*Walsh, Christopher (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. [http://www.jbc.org/content/264/5/2393.long|''Journal of biological chemistry 264: 2393 - 2396.'']
 
*Walsh, Christopher (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. [http://www.jbc.org/content/264/5/2393.long|''Journal of biological chemistry 264: 2393 - 2396.'']
 
*Wickstrum, J.R., Santangelo, T.J., and Egan, S.M. (2005) Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L-rhamnose-responsive rhaSR promoter in Escherichia coli. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251584/?report=reader|''Journal of Bacteriology 187: 6708 – 6719.''].
 
*Wickstrum, J.R., Santangelo, T.J., and Egan, S.M. (2005) Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L-rhamnose-responsive rhaSR promoter in Escherichia coli. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251584/?report=reader|''Journal of Bacteriology 187: 6708 – 6719.''].
 +
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===Biosafety Double Kill Switch function:===
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we conducted experiments to proof that the deprivation of L-Rhamnose activates the proposed Double Kill Switch system and induce cell death. E. coli with Kill Switch genes and without kill switch genes are nurtured in LB liquid culture respectively for 24 hours. From the beginning of the culturing, L-Rhamnose is added to both cultures. The culturing began at when both groups’ OD 600 equals 0.1. OD 600 is measured to indicate the concentration of bacteria body within the culture. At the 9th hour of culturing, L-Rhamnose is no longer added, and Rhamnase is added. The concentrations of control and test bacteria are compared. The result is represented in the graph: 
 +
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==Added by RDFZ-CHINA==
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[[Image: T--RDFZ-CHINA--RDFZ-1.jpg | thumb | center | 300px |Figure 1 ]]
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The result shows that the bacteria with kill switch, after a lagging time for around 3 hours after rhamnose is no longer added and rhamnase is added, began to significantly decrease in concentration, whereas the control group that carry no kill switch genes were unaffected by the deprivation of rhamnose and continue to increase in concentration. The comparison of the bacteria’s performance successfully demonstrates that the kill switch effectively kills the bacteria when L-rhamnose is not present in the environment.
  
  

Latest revision as of 16:02, 9 October 2022

Biosafety-System araCtive (AraC)

The Biosafety-System araCtive BBa_K1172909 is an improvement of the BioBrick BBa_K914014 by replacing:

  • the first promoter arabinose PBAD by the rhamnose promoter PRha to obtain a lower basal transcription.
  • intgration of the alanine racemase BBa_K1172901(alr) for gaining higher plasmid stability and for taking advantage of the double-kill switch.
  • the repressor LacI to AraC and the lac promoter to the arabinose promoter PBAD for a higher repression and lower basal transcription of the second part.

For more details about the seperate genes in this Biosafety-System and their function, click [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S here]

For more details about the function of the Biosafety-System in general, click [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System here]



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1374
    Illegal NheI site found at 2393
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1298
    Illegal BamHI site found at 2000
    Illegal BamHI site found at 2333
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1416
    Illegal AgeI site found at 1716
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1173
    Illegal BsaI.rc site found at 3068


Biosafety-System araCtive


Combining the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S genes] described above with the Biosafety-Strain K-12 ∆alr ∆dadX results in a powerful device, allowing us to control the bacterial cell division. The control of the bacterial growth is possible either active or passive. Active by inducing the PBAD promoter with L-arabinose and passive by the induction of L-rhamnose. The passive control makes it possible to control the bacterial cell division in a defined closed environment, like the MFC, by continuously adding L-rhamnose to the medium. As shown in the Figure 1 below, this leads to an expression of the essential alanine racemase (alr) and the AraC regulator, so that the expression of the RNase Ba is repressed.



Figure 1: Biosafety-System araCtive in the presence of L-rhamnose. The essential alanine racemase (Alr) and the repressor AraC are expressed, resulting in a repression of the expression of the RNAse Ba. Consequently the bacteria show normal growth behaviour.


In the event that bacteria exit the defined environment of the MFC or L-rhamnose is not added to the medium any more, both the expression of the alanine racemase (Alr) and the AraC regulatordecrease, so that the expression of the toxic RNase Ba (Barnase) begins. The cleavage of the intracellular RNA by the Barnase and the lack of synthesized D-alanine, caused by the repressed alanine racemase inhibit the cell division and makes sure that the bacteria can only grow in the defined environment or the device of choice respectively.


Figure 2: Active Biosafety-System araCtive outside of a defined environment lacking L-rhamnose. Both the expression of the alanine racemase (Alr) and AraC repressor are reduced and ideally completely shutdown. In contrast, the expression of the RNase ba (Barnase) is turned on, leading to cell death by RNA cleavage.



Results


Characterization of the arabinose promoter PBAD

First of all, the arabinose promoter PBAD was characterized to get a first impression of its basal transcription rate. Therefore the bacterial growth was investigated under the pressure of the unrepressed PBAD promoter on different carbon source using M9 minimal medium with either glucose or glycerol. The transcription rate was determined by fluorescence measurement of GFP BBa_E0040 transcribed via the PBAD promoter using the BioBrick BBa_I13541.
As shown in Figure 3 below, the bacteria grew better on glucose than on glycerol. This is caused by the fact that glucose is the better energy source of this two, because glycerol enters glycolysis at a later step and therefore delivers less energy (in addition to the additional ATP consumption to drive glycerol uptake). For the investigation of the basal transcription the fluorescence measurements, shown in Figure 4, is more interesting.
Both the wild type E. coli K-12 and the strain E. coli K-12 ∆alr ∆dadX ∆araC show about the same fluorescence on glucose (red and orange curve) but differ on glycerol. This can be explained by the fact that growth on glucose does not cause an additional induction of the arabinose promoter PBAD, while glycerol does. In the presence of glucose, the intracellular concentration of cAMP is low. The absence of cAMP results in inactive CAP (carbon utilization activator protein) and thus no induction of inefficient catabolisms like the catabolism of arabinose. Therefore, glucose is catabolized by the bacteria first, resulting in an optimal growth. In the absence of glucose, the cAMP level increases which enhances via CAP-cAMP the transcription of most operons encoding enzymes for the catabolism of alternative carbon sources. Therefore, the expression of GFP under the control of the PBAD promoter increases on glycerol. Another difference can be seen between the wild type (black curve) and the strain K-12 ∆alr ∆dadX ∆araC (blue curve). The Biosafety-Strain shows lower expression then the wild type, but has about the same growth rate, according to Figure 11. The reason for this characteristic is caused by the deleted AraC protein in the Biosafety-Strain. Because the AraC protein functions not only as are repressor but also as an activator the transcription rate decreases in the Biosafety-Strain.




Figure 3: Characterization of the bacterial growth of the strain K-12 ∆alr ∆dadX ∆araC containing the plasmid BBa_I13541 with GFP (BBa_E0040) under the control of the PBAD promoter. The M9 medium was supplemented with 5 mM D-alanine. It could be demonstrated, that the bacteria grow faster on M9 minimal medium containing glucose than on M9 minimal medium with glycerol.


Figure 4: Characterization of the fluorescence of the strain K-12 ∆alr ∆dadX ∆araC containing the plasmid BBa_I13541 with GFP (BBa_E0040) under control of the PBAD promoter. The Biosafety-Strain was cultivated on M9 minimal medium with 5 mM D-alanine supplemented.


The effect that glucose represses the transcription of the PBAD promoter becomes more obvious by calculating of the specific production rates of GFP, as shown in Figure 5. The specific production rates were calculated via equation (1) :


IGEM Bielefeld 2013 Sepzifische Produktionsrat.png


With the specific production rate of GFP, it can be demonstrated that GFP synthesis rates differ extremely between the cultivation on glucose and glycerol. The specific production rate is constantly very low when using glucose as carbon source, but shows a higher more unstable level in the cultivation with glycerol.
Because the specific production rate was calculated between every single measurement point, the curve in Figure 5 is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. And as neither of this curves are ideal, the fluctuations are the result. Nevertheless this graph shows clearly the difference between the two carbon sources.


Figure 5: Specific production rate of GFP transcribed via the PBAD promoter in dependence of different carbon sources.


Characterization of the Biosafety-System araCtive


The Biosafety-System araCtive was characterized on M9 minimal medium using glycerol as carbon source. As for the characterization of the pure arabinose promoter PBAD above, the bacterial growth and the fluorescence of GFP BBa_E0040 was measured. Therefore, the wild type and the Biosafety-Strain E. coli K-12 ∆alr ∆dadX, both containing the Biosafety-Plasmid BBa_K1172909, were cultivated once with the induction of 1% L-rhamnose and once only on glycerol.
It becomes obvious (Figure 6) that the bacteria, induced with 1 % L-rhamnose (red and black curve) grow significantly slower than on pure glycerol (orange and blue curve). This is attributed to the high metabolic burden encountered by the induced bacteria. The expression of the repressor AraC and the alanine racemase (Alr) simultaneously causes a high strain on the cells, so that they grow slower than the uninduced cells, which express only GFP.
Comparing the bacterial growth with the fluorescence in Figure 7, it can be seen that the fluorescence seems to follow the same trend than the bacterial growth. The uninduced cells show approximately an exponential rise of fluorescence, while in comparision the fluorescence of the induced bacteria increases only slowly.


Figure 6: Characterization of the bacterial growth of the Biosafety-System on M9 minimal medium with glycerol. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆alr ∆dadX containing the Biosaftey-Plasmid BBa_K1172909 and the induction by 1% L-rhamnose to pure glycerol.


Figure 7: Characterization of the fluorescence of the Biosafety-System araCtive. The Figure compares the wild type K-12 and the Biosafety-Strain K-12 ∆alr ∆dadX containing the Biosafety-Plasmid BBa_K1172909 and the induction by 1% L-rhamnose to pure glycerol..


From the data presented above it can not be determined if the expression of the repressor AraC does affect the transcription of GFP or not. The slower growth of the bacteria is a first indication that the repressor AraC and the alanine racemase (Alr) are highly expressed, but the growth of the bacteria shows nearly the same kinetics as the fluorescence. So it could be possible that the repressor does not affect the expression level of GFP under the control of the arabinose promoter PBAD. This becomes more clear by the calculation of the specific production rate of GFP by equation (1) . As shown in Figure 8 below the specific production rate differs clearly between the uninduced Biosafety-System and the Biosafety-System induced by 1% L-rhamnose. The production of GFP in the presence of L-rhamnose (red curve) is always lower than in its absence (orange curve), so that the expression of GFP is repressed in the presence of L-rhamnose.
Because the specific production rate of GFP was calculated between every single measurement point, the curve in Figure 8 is not smoothed and so the fluctuations have to be ignored, as they do not stand for are real fluctuations in the expression of GFP. They are caused by measurement variations in the growth curve and the fluorescence curve. But there is a clear tendency that the production of GFP is significantly lower when the bacteria are induced with 1% L-rhamnose. So the Biosafety-System araCtive works.


Figure 8: Specific production rate of GFP for the Biosafety-System araCtive, calculated via equation (1). The production rate of GFP of the uninduced bacteria is significantly higher compared to the bacteria induced with 1% L-rhamnose. The Biosafety-System AraCtive works.

Conclusion of the Results

For the conclusion of the result, visit our [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S#Conclusions wiki].


References

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Biosafety Double Kill Switch function:

  we conducted experiments to proof that the deprivation of L-Rhamnose activates the proposed Double Kill Switch system and induce cell death. E. coli with Kill Switch genes and without kill switch genes are nurtured in LB liquid culture respectively for 24 hours. From the beginning of the culturing, L-Rhamnose is added to both cultures. The culturing began at when both groups’ OD 600 equals 0.1. OD 600 is measured to indicate the concentration of bacteria body within the culture. At the 9th hour of culturing, L-Rhamnose is no longer added, and Rhamnase is added. The concentrations of control and test bacteria are compared. The result is represented in the graph: 

Added by RDFZ-CHINA

Figure 1

The result shows that the bacteria with kill switch, after a lagging time for around 3 hours after rhamnose is no longer added and rhamnase is added, began to significantly decrease in concentration, whereas the control group that carry no kill switch genes were unaffected by the deprivation of rhamnose and continue to increase in concentration. The comparison of the bacteria’s performance successfully demonstrates that the kill switch effectively kills the bacteria when L-rhamnose is not present in the environment.