Part:BBa_K2278011

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.