Difference between revisions of "Part:BBa K2278011"
 
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<partinfo>BBa_K2278011 short</partinfo>
 
<partinfo>BBa_K2278011 short</partinfo>
  
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<h3 id="RT"> Biological background </h3>
 
<h3 id="RT"> Biological background </h3>
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The alpha acetolactate synthase (ALS) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized into diacetyl (figure 1). This enzyme is crucial in the synthesis pathway of diacetyl in <i>Lactococcus lactic</i>.
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The alpha acetolactate synthase (Als) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized into diacetyl (figure 1). This enzyme is crucial in the synthesis pathway of diacetyl in <i>Lactococcus lactic</i>.
  
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<figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/0/0d/Als.png" width = "600" /> <figcaption> Figure 1: <b> Simplified Diacetyl pathwthay </b> : the ALS enzyme catalyzes acetolactate production, which is then spontaneously oxidized into diacetyl.
 
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</figcaption> </figure>
 
  
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As diacetyl is not produced by wild type <i>Vibrio harveyi</i>, the gene responsible for ALS production was inserted in a genetic construction downstream the pTet promoter.
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[[Image:T--INSA-UPS_France--ALSpathway.png|800px|thumb|center|'''Figure 1:''' <b>Simplified diacetyl pathway</b>: the Als enzyme catalyzes acetolactate production, which is then spontaneously oxidized into diacetyl.]]
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As diacetyl is not produced by wild type <i>Vibrio harveyi</i>, the gene responsible for Als production was 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.  
 
The pTet repressible promoter enables a compatibility with the TetR/pTet inverter system and, a constitutive transcription of the gene in absence of tetR.  
  
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<h3 id="RT"> 2- Usage in iGEM projects </h3>
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<h3 id="RT">Usage in iGEM projects </h3>
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<p> The BBa_K2278011 cames from 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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<p> The BBa_K2278011 cames from 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 establish an prokaryote-eukaryote synthetic communication. The diacetyl produced by <i>Vibrio harveyi</i> could trigger a signalling pathway in the engineered yeast <i>Pichia pastoris</i>.</p>
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It was designed to establish a prokaryote-eukaryote synthetic communication as the diacetyl produced by <i>Vibrio harveyi</i> could trigger a signalling pathway in the engineered yeast <i>Pichia pastoris</i>.</p>
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<b>Analysis of the restriction map </b>
 
<b>Analysis of the restriction map </b>
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/f/fe/GelVh3diacetyl.png" width = "400"/><figcaption> Figure 2: <b>Analysis of K2278011 in pSB1C3.  </b> BBa_K2278011 was subcloned in pSB1C3. Plasmids  
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[[Image:T--INSA-UPS_France--GelVh3diacetyl.png|800px|thumb|center|'''Figure 2:''' <b>Analysis of BBa_K2278011 in pSB1C3.  </b> BBa_K2278011 was subcloned in pSB1C3. Plasmids  
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issued from 4 different clones were analyzed to check their profiles after double digestion by EcoRI and PstI. Fragments were electrophoresed through a 0.7% agarose gel.  
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issued from 4 different clones were analyzed to check their profiles after double digestion by EcoRI and PstI. Fragments were electrophoresed through a 1% agarose gel.  
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Lane 1 is the DNA ladder (New England biolab), the 1 kb and 3kb DNA fragments are annotated. Lanes 2-5 are the digested plasmids resulting from DNA extraction of the 4 obtained clones. We expected two bands circa 2000bp and 1800 bp. </figcaption></figure> </figcaption></figure>
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Lane 1 is the DNA ladder (New England biolab), the 1.5 kb and 3kb DNA fragments are annotated. Lanes 2-5 are the digested plasmids resulting from DNA extraction of the 4 obtained clones. We expected two bands at 1930 and 2029 bp.]]
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<p><b>Sequencing </p></b>
 
<p><b>Sequencing </p></b>
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/8/8c/SeqVh3.png" width = "500"/><figcaption> Figure 3: <b>Sequencing  of the biobrick </b> 1500 ng of plasmid were sequenced. 3 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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[[Image:T--INSA-UPS_France--seqVh3.png|800px|thumb|center|'''Figure 3:''' <b>Sequencing  of the biobrick: </b> 1500 ng of plasmid were sequenced. 3 oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278011 sequence with the iGEM sequencing online tools.]]
  
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The sequencing suggests a putative deletion in the early amino acids of the coding sequence (bp #281) and a substitution (bp#437) in comparison to the expected sequence. However, only a single run was performed for this part of the sequence, which is insufficient to be affirmative about the reality of these mutations.
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<html>
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The sequencing suggested a putative deletion in the early amino acids of the coding sequence (bp #281) and a substitution (bp#437) in comparison to the expected sequence. However, only a single run was performed for this part of the sequence, which is insufficient to be affirmative about the reality of these mutations.
  
 
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<h3 id="RT"> 2- Validation of the diacetyl generator <i>in vivo</i>  </h3>
 
<h3 id="RT"> 2- Validation of the diacetyl generator <i>in vivo</i>  </h3>
  
 
<p>  
 
<p>  
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Strain <i>E. coli-ALS</i> and its control with an empty vector was grown in M9 medium supplemented with xylose (60mM) and pyruvate (40mM) as substrates. Supernatants were sampled and the tubes were plugged with parafilm to prevent diacetyl evaporation. NMR analysis was then performed to search for diacetyl production (figure 4 ; 800 MHz spectrometer from Bruker, Germany).
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Strain <i>E. coli-Als</i> and its control with an empty vector was grown in M9 medium supplemented with xylose (60mM) and pyruvate (40mM) as substrates to favorize the disponibility of diacetyl precursor. Supernatants were sampled and the tubes were plugged with parafilm to prevent diacetyl evaporation. NMR analysis was then performed to search for diacetyl production (figure 4 ; 500 MHz spectrometer from Bruker, Germany).
 
</p>  
 
</p>  
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/1/12/Diacetyl_RMN_de_kalitäy.jpg" width = "700"><figcaption> Figure 4: <b>Validation of diacetyl production by NMR analysis. </b>details of overlaid NMR spectra of <i>E. coli</i> supernatants. Green: <i>E. coli</i> pSB1C3 negative control. Purple: <i>E. coli</i> pSB1C3 + 0.50 mM diacetyl standard solution (positif control underlying the signature of diacetyl as a chemical shift of δ = 2.34 ppm). Red and blue:  <i>E. coli</i>-ALS assays.</figcaption></figure>
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[[Image:T--INSA-UPS_France--Results_5.png|800px|thumb|center|'''Figure 4:''' <b>Validation of diacetyl production by NMR analysis. </b>spectrum from 2.330 to 2.351 ppm of overlaid NMR spectra of <i>E. coli</i> supernatants. Green: <i>E. coli</i> pSB1C3 negative control. Red: <i>E. coli</i> pSB1C3 + 0.50 mM diacetyl standard solution (positive control underlying the signature of diacetyl as a chemical shift of δ = 2.34 ppm). Blue:  two replicates of <i>E. coli</i>-ALS assays.]]
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On the two E.coli (als) culture supernatants, we can observe the expected peak for diacetyl. Those results suggest a weak diacetyl production .  
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For the two <i>E. coli</i>-ALS culture supernatants, we can observe the expected peak for diacetyl. Those results suggest a weak diacetyl production.  
  
 
<p><b>Conclusion : </b> </p>
 
<p><b>Conclusion : </b> </p>
 
 
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To conclude, pSB1C3-ALS (diacetyl generator) was constructed and diacetyl production was shown by NMR. However this production is low and putative mutation observed in the ALS sequence have not been thoroughfully investigate during the course of this project.
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pSB1C3-als (diacetyl generator) was constructed and diacetyl production was shown by NMR. However this production is low and putative mutation observed in the <i>als</i> sequence have not been thoroughly investigated during the course of this project.
  
 
<p><b> Perspectives : </b> </p>
 
<p><b> Perspectives : </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 in <i>E. coli</i>, the same construction should be conjugated in <i>V. harveyi</i> to check its functionality in this background.
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Other clones were obtained for this construction and they should be sequenced before trying again this experiment. The same construction should then be conjugated in <i>V. harveyi</i> to check its functionality in this background.
  
 
</html>
 
</html>
  
 
===Design Notes===
 
===Design Notes===
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pTet : BBa_40040 is mutated : substitution at the 43th pair (G-> T) due to IDT complexity requirements for gBlocks  synthesis. The effect of the mutation was not investigated.
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pTet : BBa_40040* is mutated : substitution at the 43th pair (G-> T) due to IDT complexity requirements for gBlocks  synthesis. The effect of the mutation was not investigated.
  
 
Terminator : BBa_B1006* is mutated : the initial A at the 35th position is substituted by a T due to IDT complexity requirements for gBlocks synthesis.
 
Terminator : BBa_B1006* is mutated : the initial A at the 35th position is substituted by a T due to IDT complexity requirements for gBlocks synthesis.
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===Source===
 
===Source===
  
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This enzyme cames from Lactococcus lactis subsp. lactis Il1403 DNA genomic sequence.
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This enzyme cames from <i>Lactococcus lactis</i> subsp. lactis Il1403 DNA genomic sequence.
 
Sequence available at https://www.ncbi.nlm.nih.gov/gene/1114827
 
Sequence available at https://www.ncbi.nlm.nih.gov/gene/1114827
  

Latest revision as of 09:31, 31 October 2017

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 conditionally produce diacetyl in a Vibrio harveyi strain.

Biological background

The alpha acetolactate synthase (Als) enzyme catalyzes the conversion of pyruvate to acetolactate, which is then spontaneously oxidized into diacetyl (figure 1). This enzyme is crucial in the synthesis pathway of diacetyl in Lactococcus lactic.

Figure 1: Simplified diacetyl pathway: the Als enzyme catalyzes acetolactate production, which is then spontaneously oxidized into diacetyl.

As diacetyl is not produced by wild type Vibrio harveyi, the gene responsible for Als production was 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.

Usage in iGEM projects

The BBa_K2278011 cames from the Croc’n cholera project (team INSA-UPS-France 2017). It was designed to establish a prokaryote-eukaryote synthetic communication as the diacetyl produced by Vibrio harveyi could trigger a signalling pathway in the engineered yeast Pichia pastoris.

Experiments

1- Molecular biology

The gene was placed in silico under the control of the pTet promoter (BBa_R0040), 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 the pSB1C3 plasmid and transformed into E. coli Dh5-alpha strain. 5 transformants were obtained.

Analysis of the restriction map

Figure 2: Analysis of BBa_K2278011 in pSB1C3. BBa_K2278011 was subcloned in pSB1C3. Plasmids issued from 4 different clones were analyzed to check their profiles after double digestion by EcoRI and PstI. Fragments were electrophoresed through a 1% agarose gel. Lane 1 is the DNA ladder (New England biolab), the 1.5 kb and 3kb DNA fragments are annotated. Lanes 2-5 are the digested plasmids resulting from DNA extraction of the 4 obtained clones. We expected two bands at 1930 and 2029 bp.


Sequencing

Figure 3: Sequencing of the biobrick: 1500 ng of plasmid were sequenced. 3 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 suggested a putative deletion in the early amino acids of the coding sequence (bp #281) and a substitution (bp#437) in comparison to the expected sequence. However, only a single run was performed for this part of the sequence, which is insufficient to be affirmative about the reality of these mutations.

2- Validation of the diacetyl generator in vivo

Strain E. coli-Als and its control with an empty vector was grown in M9 medium supplemented with xylose (60mM) and pyruvate (40mM) as substrates to favorize the disponibility of diacetyl precursor. Supernatants were sampled and the tubes were plugged with parafilm to prevent diacetyl evaporation. NMR analysis was then performed to search for diacetyl production (figure 4 ; 500 MHz spectrometer from Bruker, Germany).

Figure 4: Validation of diacetyl production by NMR analysis. spectrum from 2.330 to 2.351 ppm of overlaid NMR spectra of E. coli supernatants. Green: E. coli pSB1C3 negative control. Red: E. coli pSB1C3 + 0.50 mM diacetyl standard solution (positive control underlying the signature of diacetyl as a chemical shift of δ = 2.34 ppm). Blue: two replicates of E. coli-ALS assays.

For the two E. coli-ALS culture supernatants, we can observe the expected peak for diacetyl. Those results suggest a weak diacetyl production.

Conclusion :

pSB1C3-als (diacetyl generator) was constructed and diacetyl production was shown by NMR. However this production is low and putative mutation observed in the als sequence have not been thoroughly investigated during the course of this project.

Perspectives :

Other clones were obtained for this construction and they should be sequenced before trying again this experiment. The same construction should then be conjugated in V. harveyi to check its functionality in this background.

Design Notes

pTet : BBa_40040* is mutated : substitution at the 43th pair (G-> T) due to IDT complexity requirements for gBlocks synthesis. The effect of the mutation was not investigated.

Terminator : BBa_B1006* is mutated : the initial A at the 35th position is substituted by a T due to IDT complexity requirements for gBlocks synthesis.

Source

This enzyme cames from Lactococcus lactis subsp. lactis Il1403 DNA genomic sequence. Sequence available at https://www.ncbi.nlm.nih.gov/gene/1114827

References

Marugg, J. D., D. Goelling, U. Stahl, A. M. Ledeboer, M. Y. Toonen, W. M. Verhue, and C. T. Verrips. 1994. Identification and characterization of the α-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis. Appl. Environ. Microbiol. 60:1390-1394.

Hugenholtz, J., Kleerebezem, M., Starrenburg, M., Delcour, J., de Vos, W. and Hols, P. (2000). Lactococcus lactis as a Cell Factory for High-Level Diacetyl Production. Applied and Environmental Microbiology, 66(9), pp.4112-4114.