Difference between revisions of "Part:BBa K1172905"
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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>
 
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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Moreover the BioBrick is not limited to this Biosafety-System, as it can be used for the regulated trancription of any coding sequence behind the P<sub>''BAD''</sub> promoter, opening the possibility of an antibiotic-free selection in the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain Biosafety-Strain].
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Moreover the BioBrick is not limited to this Biosafety-System, as it can be used for the regulated trancription of any coding sequence behind the P<sub>''BAD''</sub> promoter, opening the possibility of an antibiotic-free selection in the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain Biosafety-Strain].</p><br>
  
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K1172905 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1172905 SequenceAndFeatures</partinfo>
  
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==='''Characterization of the arabinose promoter pBAD'''===
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==='''Biosafety-System araCtive'''===
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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>
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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>
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[[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.]]
 
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[[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.]]
 
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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>
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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>
  
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GLEICHUNG s p rate<br>
 
  
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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>
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[[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.]]
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[[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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==='''Characterization of the Biosafety-System araCtive'''===
 
 
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<p align="justify">
 
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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.
 
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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>
 
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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>
 
  
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[[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.]]
 
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[[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.]]
 
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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>
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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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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>
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[[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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[[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.]]
 
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==='''Conclusion of the Results'''===
 
 
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<p align="justify">
 
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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>
 
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[[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).]]
 
  
  

Revision as of 15:23, 30 October 2013

Part 1 of the Biosafety-System araCtive


Usage and Biology

These biobrick is the first part of the Biosafety-System AraCtive BBa_K1172909, which 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 mechanism.
  • the repressor LacI BBa_C0012 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]</p>

Moreover the BioBrick is not limited to this Biosafety-System, as it can be used for the regulated trancription of any coding sequence behind the PBAD promoter, opening the possibility of an antibiotic-free selection in the [http://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_Strain Biosafety-Strain].</p>

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1374
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1298
    Illegal BamHI site found at 2000
  • 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


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.



References

  • Baldoma L and Aguilar J (1988) Metabolism of L-Fucose and L-Rhamnose in Escherichia coli: Aerobic-Anaerobic Regulation of L-Lactaldehyde Dissimilation [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210658/pdf/jbacter00179-0434.pdf|Journal of Bacteriology 170: 416 - 421.].
  • Carafa, Yves d'Aubenton Brody, Edward and Claude (1990) Thermest Prediction of Rho-independent Escherichia coli Transcription Terminators - A Statistical Analysis of their RNA Stem-Loop Structures [http://ac.els-cdn.com/S0022283699800059/1-s2.0-S0022283699800059-main.pdf?_tid=ede07e2a-2a92-11e3-b889-00000aab0f6c&acdnat=1380629809_2d1a59e395fc69c8608ab8b5aea842f7|Journal of molecular biology 216: 835 - 858].
  • Cass, Laura G. and Wilcoy, Gary (1988) Novel Activation of araC Expression and a DNA Site Required for araC Autoregulation in Escherichia coli B/r [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC211425/pdf/jbacter00187-0394.pdf|Journal of Bacteriology 170: 4174 - 4180].
  • Hamilton, Eileen P. and Lee, Nancy (1988) Three binding sites for AraC protein are required for autoregulation of araC in Escherichia coli [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC279856/pdf/pnas00258-0029.pdf|Proc Natl Acad Sci U S A 85: 1749 - 53].
  • Mossakowska, Danuta E. Nyberg, Kerstin and Fersht, Alan R. (1989) Kinetic Characterization of the Recombinant Ribonuclease from Bacillus amyloliquefaciens (Barnase) and Investigation of Key Residues in Catalysis by Site-Directed Mutagenesis [http://pubs.acs.org/doi/pdf/10.1021/bi00435a033|Biochemistry 28: 3843 - 3850.].
  • Paddon, C. J. Vasantha, N. and Hartley, R. W. (1989) Translation and Processing of Bacillus amyloliquefaciens Extracellular Rnase [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC209718/pdf/jbacter00168-0575.pdf|Journal of Bacteriology 171: 1185 - 1187.].
  • Schleif, Robert (2010) AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action [http://gene.bio.jhu.edu/Ourspdf/127.pdf|FEMS microbial reviews 34: 779 - 796.].
  • Voss, Carsten Lindau, Dennis and Flaschel, Erwin (2006) Production of Recombinant RNase Ba and Its Application in Downstream Processing of Plasmid DNA for Pharmaceutical Use [http://onlinelibrary.wiley.com/doi/10.1021/bp050417e/pdf|Biotechnology Progress 22: 737 - 744.].
  • 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.].