Difference between revisions of "Part:BBa K2278011"
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<p><b>sous titre</b></p>
 
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<p> Protocole </p>
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Strain E. coli-ALS and its control with an empty vector was grown in M9 medium and supernatant was sampled at XX. Traitement du surnageant ? Samples were analyze by Mass Spectrometry and NMR (?).
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The tubes are plugged with parafilm to prevent dactyl evaporation.
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A control is performed with a tube of E. coli TOP10 (pSB1C3) and a tube without parafilm. A solution of 500uM of diacetyl is prepared as standard for calibration
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500 μL of each solution and 100 μL TSP-d4 as internal standard and reference was added into a NMR tube. Samples are analyzed thanks to a 500 MHz NMR spectrometer Brucker advance 500
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<h3 id="RT">1- Validation of </h3>
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<h3 id="RT">1- Validation of the dactyl generator</h3>
 
description  
 
description  
  
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<figure><p style="text-align:center;"><img src="" width = "500"/><figcaption> Figure  <b>title </b>légende </figcaption></figure>
 
<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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<p><b>Conclusion : </b> </p>
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To conclude, pSB1C3-3 (diacetyl generator) was successfully constructed by molecular biology: its length and restriction map were confirmed by restriction analyses. The diacetyl production was quantified and detected by NMR. No significant results were obtained.
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This can be likely related to a mutation observed in the ALS sequence of the investigated clone.
  
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<h3 id="RT">2. 2ème approche  </h3>
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The diacetyl from the commercial standard was successfully detected and quantified. This leads to the validation of a RMN based method to prepare sample, detect and quantify the diacetyl. This method shows a good sensitivity as it can detect down to 25nm of diacetyl in the samples.  
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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> Perspectives : </b> </p>
  
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<p><b>Discussion : </b> </p>
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Other clones were obtained for this construction and they should be sequenced before trying again this experiment. Once diacetyl production is validated, the same construction should be conjugated in V. harveyi to check its functionality in this background.
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<p>des perspectives éclectiques </p>
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Revision as of 04:23, 16 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

Strain E. coli-ALS and its control with an empty vector was grown in M9 medium and supernatant was sampled at XX. Traitement du surnageant ? Samples were analyze by Mass Spectrometry and NMR (?). The tubes are plugged with parafilm to prevent dactyl evaporation. A control is performed with a tube of E. coli TOP10 (pSB1C3) and a tube without parafilm. A solution of 500uM of diacetyl is prepared as standard for calibration 500 μL of each solution and 100 μL TSP-d4 as internal standard and reference was added into a NMR tube. Samples are analyzed thanks to a 500 MHz NMR spectrometer Brucker advance 500

Characterization

1- Validation of the dactyl generator

description

manip1

Image stylée

Figure title légende

Conclusion :

To conclude, pSB1C3-3 (diacetyl generator) was successfully constructed by molecular biology: its length and restriction map were confirmed by restriction analyses. The diacetyl production was quantified and detected by NMR. No significant results were obtained. This can be likely related to a mutation observed in the ALS sequence of the investigated clone. The diacetyl from the commercial standard was successfully detected and quantified. This leads to the validation of a RMN based method to prepare sample, detect and quantify the diacetyl. This method shows a good sensitivity as it can detect down to 25nm of diacetyl in the samples.

Perspectives :

Other clones were obtained for this construction and they should be sequenced before trying again this experiment. Once diacetyl production is validated, the same construction should be conjugated in V. harveyi to check its functionality in this background.