Difference between revisions of "Part:BBa K2278011"
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The alpha acetolactate synthase (ALS) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized as diacetyl. This enzyme is crucial in the synthesis pathway of diacetyl in Lactococcus lactic.
 
The alpha acetolactate synthase (ALS) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized as diacetyl. This enzyme is crucial in the synthesis pathway of diacetyl in Lactococcus lactic.
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In the INSA UPS France 2017 project, this part is used to establish an eukaryote-prokaryote communication system based on diacetyl so that Vibrio harveyi could trigger an engineered production pathway of the Yeast Pichia pastoris. &#8232;
 
In the INSA UPS France 2017 project, this part is used to establish an eukaryote-prokaryote communication system based on diacetyl so that Vibrio harveyi could trigger an engineered production pathway of the Yeast Pichia pastoris. &#8232;
  
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===Usage and Biology===
 
  
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<partinfo>BBa_K2278011 short</partinfo>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2278011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2278011 SequenceAndFeatures</partinfo>
  
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=='''Introduction'''==
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<html>
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This DNA biobrick was designed in order to produce in <i></i> strain.
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<h3 id="RT"> 1- Biological background </h3>
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Mécanisme
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<figure><p style="text-align:center;"> <img src ="g" width = "600" /> <figcaption> Figure 1: <btitre </b> figure caption</figcaption> </figure>
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<h3 id="RT"> 2- Usage in iGEM projects </h3>
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<p> The BBa_K2278011 cames from the module of the Croc’n cholera project <a href="http://2017.igem.org/Team:INSA-UPS_France">(team INSA-UPS-France 2017)</a>
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It was designed to produce </p>
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<p>The part includes  </p>
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</html>
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=='''Experiments'''==
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<html>
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<h3 id="RT"> 1- Molecular biology </h3>
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<p>
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The  gene was placed in silico under the control of the p promoter (BBa_R), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. 
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The construct was cloned by conventional ligation into pSB1C3 plasmid and transformed into E. coli Dh5 alpha strain. X transformants were obtained.
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</p>
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<b>Analysis of the restriction map </b>
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<figure><p style="text-align:center;"><img src="" width = "600"/><figcaption> Figure 2: <b>title  </b> Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are in parentheses. pSB1C3 (2029bp the other band correspond to a xxx bp insert)</figcaption></figure>
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<p><b>Sequencing </p></b>
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure 3: <b>Sequencing  of pSB1C3_  </b> 1500 ng of plasmid are sequenced. X oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278011 sequence with the iGEM sequencing online tools. </figcaption></figure>
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The sequencing show a
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<h3 id="RT"> 2- Expression  <i>in vivo</i>  </h3>
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<p><b>sous titre</b></p>
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<p> Protocole </p>
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</html>
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=='''Characterization'''==
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<html>
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<h3 id="RT">1- Validation of  </h3>
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description
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<p> <b>manip1 </b> </p>
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Image stylée
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure  <b>title </b>légende </figcaption></figure>
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<p>Interprétation  </p>
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<p> <b>manip2</b> </p>
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Image stylée
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure  <b>plus de figure !  </figcaption></figure>
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<p>interprétation </p>
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<p><b>Discussion : </b> </p>
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<p></p>
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<h3 id="RT">2. 2ème approche  </h3>
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure <b>Solid results </b>  légende de qualité </figcaption></figure>
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<p>brillante analyse</p>
  
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<p><b>Discussion : </b> </p>
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===Functional Parameters===
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<p>des perspectives éclectiques </p>
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<partinfo>BBa_K2278011 parameters</partinfo>
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Revision as of 11:54, 10 October 2017



The alpha acetolactate synthase (ALS) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized as diacetyl. This enzyme is crucial in the synthesis pathway of diacetyl in Lactococcus lactic. As diacetyl is not produced by wild type Vibrio harveyi, the gene responsible for ALS production is inserted in a genetic construction downstream the pTet promoter. The pTet repressible promoter enables a compatibility with the TetR/pTet inverter system and a constitutive transcription of the gene in absence of tetR. In the INSA UPS France 2017 project, this part is used to establish an eukaryote-prokaryote communication system based on diacetyl so that Vibrio harveyi could trigger an engineered production pathway of the Yeast Pichia pastoris. 



pTet driven Diacetyl generator (pTet + ALS)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1738
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This DNA biobrick was designed in order to produce in strain.

1- Biological background

Mécanisme

Figure 1: figure caption

2- Usage in iGEM projects

The BBa_K2278011 cames from the module of the Croc’n cholera project (team INSA-UPS-France 2017) It was designed to produce

The part includes


Experiments

1- Molecular biology

The gene was placed in silico under the control of the p promoter (BBa_R), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by conventional ligation into pSB1C3 plasmid and transformed into E. coli Dh5 alpha strain. X transformants were obtained.

Analysis of the restriction map

Figure 2: title Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are in parentheses. pSB1C3 (2029bp the other band correspond to a xxx bp insert)

Sequencing

Figure 3: Sequencing of pSB1C3_ 1500 ng of plasmid are sequenced. X oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278011 sequence with the iGEM sequencing online tools.
The sequencing show a

2- Expression in vivo

sous titre

Protocole

Characterization

1- Validation of

description

manip1

Image stylée

Figure title légende

Interprétation

manip2

Image stylée

Figure plus de figure !

interprétation

Discussion :

2. 2ème approche

Figure Solid results légende de qualité

brillante analyse

Discussion :

des perspectives éclectiques