Team:LMU-Munich/Bacillus BioBricks

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[[File:Bacillus BioBrick Box banner.resized WORDS.JPG|620px|link=]]
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[[Image:BacillusBioBrickBox.png|150px|right]]
 
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==Bacillus BioBricks==
 
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We will create a toolbox of <i>Bacillus</i> BioBricks to contribute to the registry.
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[[Image:BacillusBioBrickBox.png|100px|right|link=]]
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'''We would like to introduce ''Bacillus'' to the world of iGEM!!!'''
 
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[[Image:LMU-Munich_Introduce_Bacillus.jpg|400px|center]]
 
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=='''B<sup>4</sup>''' - 22 core parts for ''Bacillus subtilis''==
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<br>
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<p align="justify">A major goal of our iGEM project is to [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction introduce ''B. subtilis''] as a new chassis for BioBrick-based synthetic biology. For that purpose, we created a toolbox of <i>Bacillus</i> BioBricks to contribute to the registry to make it accessible to many more future iGEM-teams and the entire public microbiology domain! This ''<b>Bacillus'' B</b>io<b>B</b>rick <b>B</b>ox ('''B<sup>4</sup>''') contains the following ''Bacillus'' specific parts:</p>
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This <b>B</b>acillus <b>B</b>io<b>B</b>rick <b>B</b>ox contains Bacillus specific:
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<div class="box">
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{| width="100%"
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks/Vectors|Vectors]]'''
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{|
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks/Promoters|Promoters]]'''
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|<font color="#FFFFFF">+--+--+--+--+--</font>
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks/Reporters|Reporters]]'''
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|<font color="#FFFFFF">+--+--+--+--+--</font>
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| style="width:20%"|'''[[Team:LMU-Munich/Bacillus_BioBricks/Tags|Affinity tags]]
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|<font color="#FFFFFF">+--+--+--+--+--</font>
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|-
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|[[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]
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|[[File:LMU PromoterIconBC.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters]]
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|[[File:LMU Reporter.png|60px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters]]
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|[[File:Proteinaffinitytagbutton.png|50px|link=Team:LMU-Munich/Bacillus_BioBricks#Affinity_Tags]]
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|-
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|valign="top"|<font size="2">
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pSB<sub>''Bs''</sub>1C<br>
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pSB<sub>''Bs''</sub>4S<br>
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pSB<sub>''Bs''</sub>2E<br>
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pSB<sub>''Bs''</sub>1C-''lac''Z<br>
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pSB<sub>''Bs''</sub>3C-''lux''ABCDE<br>
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pSB<sub>''Bs''</sub>4S-P<sub>''xyl''</sub><br>
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pSB<sub>''Bs''</sub>0K-P<sub>''spac''</sub><br>
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'''Sporo'''vector</font>
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|valign="top"|<font size="2">
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Anderson<br>
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P<sub>''liaG''</sub><br>
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P<sub>''veg''</sub><br>
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P<sub>''lepA''</sub><br>
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P<sub>''liaI''</sub><br>
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''xylR''-P<sub>''xyl''</sub></font>
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|valign="top"|<font size="2">
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''gfp''<br>
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''mkate2''<br>
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''lacZ''<br>
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''luc+''<br></font>
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|valign="top"|<font size="2">
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Flag<br>His<br>cMyc<br>Strep<br>HA</font>
|-
|-
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|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]'''
 
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|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Promoters|Promoter]]'''
 
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|'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Reporters|Reporter]]'''
 
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|}
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</div>
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[[File:NEXT.png|right|80px|link=Team:LMU-Munich/Germination_Stop]]  [[File:BACK.png|left|80px|link=Team:LMU-Munich/Data/differentiation_tour]]
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==Bacillus Vectors==
 
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Here is a list of all the vectors we cloned and used. Please note that pMAD is not in a BioBrick standard, but it is a useful tool to knock out genes inBacillus subtilis.
 
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For the use of our vectors, please see our [[Team:LMU-Munich/Lab_Notebook/Protocols|Protocols]] page. A general introduction to Bacillus subtilis and its integrative vectors can be found [[Team:LMU-Munich/Bacillus Introduction|here]]. All vectors have ampicillin as <i>E. coli</i> resistance and RFP as selection marker.
 
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{|
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|-
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|<b>name</b>
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|<b>E. coli res.</b>
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|<b>B. subt. res.</b>
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<p align="justify">Since ''Bacillus subtilis'' is not an organism commonly used in iGEM, please check out our [https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction Introduction] to learn more about it.</p>
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|<b>insertion locus</b>
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|<b>description</b>
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<div class="box">
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|<b>derived from</b>
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==''Bacillus'' Vectors  [[File:LMU Backbone.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks#Bacillus_Vectors|Vectors]]==
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|<b>reference</b>
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{| "width=100%" style="text-align:center;" style="align:right"|
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|-
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|<p align="justify">We have generated a suite of BioBrick-compatible vectors: three empty insertional backbones with different antibiotic resistances and integration loci, two reporter and two expression vectors.</p>  
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|<b>pSB<sub>Bs</sub>1C-<i>lacZ</i> </b>
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|[[File:LMU-Munich-PSBBs1C.png|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Vectors]]
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|Amp
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|Cam
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|amyE
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|with <i>lacZ</i> reporter gene
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|pAC6
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|?
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|-
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|<b>pSB<sub>Bs</sub>4S </b>
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|Amp
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|Spec
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|thrC
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|empty
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|pDG1731
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|?
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|-
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|<b>pSB<sub>Bs</sub>1C </b>
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|Amp
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|Cam
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|amyE
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|empty
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|pDG1662
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|?
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|-
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|<b>pSB<sub>Bs</sub>4S-P<sub><i>Xyl</i></sub> </b>
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|Amp
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|Spec
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|thrC
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|with Xylose-inducible promoter
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|pXT
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|?
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|<b>pSB<sub>Bs</sub>3C-<i>luxABCDE</i> </b>
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|Amp
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|Cam
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|sacA
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|with <i>luxABCDE</i> reporter cassette
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|pAH328
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|?
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|-
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|<b>pSB<sub>Bs</sub>0K-P<sub><i>spac</i></sub> </b>
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|Amp
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|Kan
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|replicative
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|with IPTG inducible promoter
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|pDG148
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|?
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|-
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|<b>pSB<sub>Bs</sub>2E </b>
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|Amp
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|MLS
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|lacA
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|empty
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|pAX01
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|?
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Bacillus BioBricks/Vectors]]
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</div>
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The number in the vector's name codes for the insertion locus and the following letter for the <i> Bacillus subtilis </i> resistance gene according to the following table:
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<div class="box">
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==''Bacillus'' Promoters  [[File:LMU PromoterIconBC.png|100px]]==
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{|
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{| "width=100%" style="align:right"|
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|-
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|<b>number</b>
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|<b>insertion locus</b>
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|<b>letter</b>
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|<b>resistance </b>
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|-
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|<b>0</b>
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|replicative
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|<b>C</b>
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|Chloramphenicol
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|<b>1</b>
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|amyE (amylase)
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|<b>E</b>
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|MLS (Erythromycin + Lincomycin)
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|<b>2</b>
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|lacA (laccase ?)
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|<b>K</b>
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|Kanamycin
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|<b>3</b>
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|sacA (saccase?)
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|<b>S</b>
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|Spectinomycin
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|<b>4</b>
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|thrC (threonine C)
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<p align="justify">To provide a set of promoters of different strength we characterized several promoters in ''Bacillus subtilis''. Both constitutive and inducible promoters are covered.</p>
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[[File:Promoters overview.png|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Promoters]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Bacillus BioBricks/Promoters]]
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|}
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</div>
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<div class="box">
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==''Bacillus'' Reporters  [[File:LMU Reporter.png|50px]]==
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{| "width=100%" style="align:right"|
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|
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<p align="justify">We designed and codon-optimized a set of reporters that are commonly used in ''B. subtilis''.</p>
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|
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[[File:LMU GFP.jpg|200px|right|link=Team:LMU-Munich/Bacillus BioBricks/Reporters]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Bacillus BioBricks/Reporters]]
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</div>
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The concentrations of the antibiotics and the insertion tests can be found in our protocol section.
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<div class="box">
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==Affinity Tags  [[File:Proteinaffinitytagbutton.png|50px]]==
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The promoters we submit:
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{| "width=100%" style="align:right"|
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|
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Pveg, PliaI, PliaG, plepA, Pxyl, Pxyl+XylR
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<p align="justify">We synthesized 5 affinity tags for protein purification. They all are designed in Freiburg standard with an optimized ribosome binding site upstream. We have not yet tested our tags.</p>
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We also tested the Anderson promoter collection in <i> Bacillus subtilis </i> with pSB<sub>Bs</sub>3C-<i>luxABCDE</i>. The data will be online in our data section soon.
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Furthermore we plan to submit reporter genes optimized for <i> Bacillus subtilis </i>, namely GFP, lacZ, luc and mKate.
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Useful for the registry are also 5 tages in Freiburg Standard (cMyc, 10His, Flag, Strep, HA).
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More details to come soon.
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==Bacillus Promoters==
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Another goal is to produce promoters of ''Bacillus subtilis'' in BioBrick mode and to evaluate them. These well-defined promoters will then be part of the ''Bacillus'' BioBrickBox which we will send to the registry but they can also be useful in our project '''Bead'''zillus to express our fusion crust proteins on the outside of our spores. Therefore we will use different promoters which are the constitutive promoters from the Anderson collection from the partsregistry, the constitutive promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis'' as well as the inducible promoter P<sub>''liaI''</sub> from ''B. subtilis''. For the characterization of the different promoters we will clone them upstream of reporter genes (lux operon, lacZ) to measure their activity in ''B. subtilis''. Therefore we use e.g. the two reporter vectors from ''B. subtilis'' which are also part of the ''Bacillus'' BioBrickBox. One of the reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> contains the ''lux'' operon as a reporter. This is why the activity of the promoter can be measured as luminescence with the plate reader (BioTek) which is a result of the expression of the luciferase. The second reporter vector used for the evaluation of the promoters is pSB<sub>Bs</sub>1C-''lacZ'' which contains the reporter gene ''lacZ''. The promoter activity leads to the production of the enzyme beta-galactosidase which cleaves the substrate ONPG. The product is detectable with the photometer and refers to the promoter activity.
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==Bacillus Reporters==
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- GFP
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- mKate
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- LacZ
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- luc
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'''Project Navigation'''
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Bacillus BioBricks/Tags]]
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<html>
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction">
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<img src="https://static.igem.org/mediawiki/2012/8/8d/Bacilluss_Intro.png" height=70"/>
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction"><font size="1" face="verdana">Bacillus<BR>Intro</font></a>
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====Project Navigation====
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|[[File:Bacilluss_Intro.png|100px|link=Team:LMU-Munich/Bacillus_Introduction]]
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|[[File:BacillusBioBrickBox.png|100px|link=Team:LMU-Munich/Bacillus_BioBricks]]
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|[[File:SporeCoat.png|100px|link=Team:LMU-Munich/Spore_Coat_Proteins]]
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|[[File:GerminationSTOP.png|100px|link=Team:LMU-Munich/Germination_Stop]]
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|[[Team:LMU-Munich/Bacillus_Introduction|<font size="2">'''''Bacillus'''''<BR>Intro</font>]]
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|[[Team:LMU-Munich/Bacillus_BioBricks|<font size="2" face="verdana">'''''Bacillus'''''<BR>'''B'''io'''B'''rick'''B'''ox</font>]]
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|[[Team:LMU-Munich/Spore_Coat_Proteins|<font size="2" face="verdana">'''Sporo'''beads</font>]]
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<a href="https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks"><font size="1" face="verdana">Bacillus<BR>BioBrickBOX</font></a>
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|[[Team:LMU-Munich/Germination_Stop|<font size="2" face="verdana">'''Germination'''<BR>STOP</font>]]
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Latest revision as of 16:41, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

Team-LMU culture tubes.resized.jpg

The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

IGEM HQ LMU prize.jpg

[ more news ]

Sporenfreunde

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