Difference between revisions of "Part:BBa K2278011"

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	<b>Analysis of the restriction map </b>		<b>Analysis of the restriction map </b>
−	<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/2/24/Vh3_gel.png" width = "300"/><figcaption> Figure 2: <b>Analysis of K2278011 in pSB1C3 restriction map  </b> 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) </figcaption></figure>	+	<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/2/24/Vh3_gel.png" width = "300"/><figcaption> Figure 2: <b>Analysis of K2278011 in pSB1C3 restriction map.   </b> BBa_K2278011 was cloned in pSB1C3. The plasmids of 4 obtained clones were analyzed to check their length. Double digested (EcoRI and PstI) plasmids are electrophoresed through a 0.7% agarose gel.
		+	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 expect two bands circa 2000bp and 1800 bp. </figcaption></figure>) </figcaption></figure>
	<p><b>Sequencing </p></b>		<p><b>Sequencing </p></b>

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Revision as of 11:34, 18 October 2017

pTet driven Diacetyl generator (pTet + ALS)

Sequence and Features

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 map

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Sequencing

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.

On the two E.coli (als) culture supernatants, we can clearly observed a signal at δ = 2.38 ppm, corresponding to the same chemical shift of the diacetyl spiked assay. The diacetyl 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.