Part:BBa_K2278011
pTet driven Diacetyl generator (pTet + ALS)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1738
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
This DNA biobrick was designed in order to produce conditionally diacetyl in Vibrio harveyi strain.
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. Mecanisme 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 mapSequencing
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
Validation of the diacetyl generator
A production assay of diacetyl is performed.Validation of diacetyl production by NMR analysis
On the two E.coli (als) culture supernatants, we can clearly observed a signal at d = 2.38 ppm, corresponding to the same chemical shift of the diacetyl spiked assay. This peak is not visible on the negative control (green). Those results strongly suggest a diacetyl production which has to be confirmed by additional NMR experiments.Conclusion :
To conclude, pSB1C3-3 (diacetyl generator) was successfully constructed by molecular biology: its length and restriction map were confirmed by restriction analyses. Results were obtained for the diacetyl production by NMR detection but has to be confirmed because of the 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 (experience not shown).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. 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.
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