Difference between revisions of "Part:BBa K2278011"
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Strain E. coli-ALS and its control with an empty vector was grown in M9 medium supplemented with xylose (60mM) and pyruvate (40mM), the tubes were plugged with parafilm to prevent diacetyl evaporation.
 
Strain E. coli-ALS and its control with an empty vector was grown in M9 medium supplemented with xylose (60mM) and pyruvate (40mM), the tubes were plugged with parafilm to prevent diacetyl evaporation.
 
The cells were pelleted to gather the supernatant to analyze it by NMR. A solution of 0.50mM of diacetyl is prepared as standard for calibration
 
The cells were pelleted to gather the supernatant to analyze it by NMR. A solution of 0.50mM of diacetyl is prepared as standard for calibration

Revision as of 22:52, 17 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 produce conditionally diacetyl in Vibrio harveyi strain.

1- Biological background

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. Mécanisme

Figure 1: Simplified Diacetyl pathwthay : The ALS enzyme catalyzes acetolactate production, which is then spontaneously oxidized as diacetyl
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.

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 establish an eukaryote-prokaryote communication system based on diacetyl so that Vibrio harveyi could trigger an engineered production pathway of the Yeast Pichia pastoris.

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. 5 transformants were obtained.

Analysis of the restriction map

Figure 2: Analysis of K2278011 in pSB1C3 restriction map Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired DNA fragments lengths are in parentheses pSB1C3(2029 bp) and BBa_K2278011 with a spacer (1930 bp)

Sequencing

Figure 3: Sequencing of pSB1C3_ 1500 ng of plasmid are 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 revealed a deletion in the early amino acids of the coding sequence (bp #281) and a substitution (bp#437) in comparison of the expected sequence. As a result the final protein is deleted of the 44th first amino acids and this might affect the activity of the enzyme.

2- Expression 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), the tubes were plugged with parafilm to prevent diacetyl evaporation. The cells were pelleted to gather the supernatant to analyze it by NMR. A solution of 0.50mM of diacetyl is prepared as standard for calibration

Characterization

1- Validation of the diacetyl generator

A production assay of diacetyl is performed.

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. No significant results were obtained for the diacetyl production by NMR detection and quantification. 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.

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.

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.

sequence available at https://www.ncbi.nlm.nih.gov/gene/1114827