Team:Toulouse/Result/parts/Submitted parts

This part is designed to enable a N-acetylglucosamine based chemotaxis in B. subtilis.

Design

The encoded protein is a chimera of two proteins:
- the methyl accepting Chemotaxis protein (McpA from B. subtilis which is required for taxis towards glucose.
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from Vibrio cholerae (VCD).

The chimeric protein contains the intracellular domains of McpA from Bacillus subtilis to enable the transduction of the signal: these domains correspond to the amino acids 1-37 and 282-610. The extracellular domain of VCD is inserted between the intracellular regions of McpA to sense N-acetylglucosamine and corresponds to the amino acids 31-310.

Type

Translation unit

Tests

This part is not tested yet.

References

Uniprot:
- McpA
- N-acetylglucosamine regulated methyl-accepting chemotaxis protein

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This part is designed to enable a N-acetylglucosamine based chemotaxis in Bacillus subtilis.

Design

This expression cassette is designed for the expression of an antifungal peptide, D4E1 and for its secretion in B. subtilis. It is composed of the strong, constitutive promoter of B. subtilis P_veg (BBa_K823003), strong RBS for B. subtilis (BBa_K780002), the open reading frame of D4E1 and a double terminator (BBa_B0015)

Type

Generator

Tests

This part was not tested yet.

References

Uniprot:
- McpA
- N-acetylglucosamine regulated methyl-accepting chemotaxis protein

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This part is designed to enable the binding of Bacillus subtilis to the fungi wall made of chitin.

Design

This part is composed of a Cell Wall Binding (CWB) sequence and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.

The CWB domain has been extracted from LytC (BBa_K316030) of the iGEM 2010 Imperial College team.
The CBD is composed of the Domain 4 of the GbpA protein of Vibrio cholerae, a protein reported to mediate bacterial attachment to chitin. It has been shown that the Domain 4 was essential to bind chitin.

Type

Generator

Tests

This part was tested with chitin beads. (See Binding module)

References

- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373

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The Gastrodia anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.

Design

This part is composed of a Strong RBS (BBa_K780002) and the open reading frame of the Gastrodia anti-fungal protein 1 (GAFP-1) optimized for its expression and its secretion in Bacillus subtilis. The codon optimization was made thanks to the DNA 2.0 software program.

Type

Translational unit

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373

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The Gastrodia anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.

Design

This part is composed of a Strong RBS (BBa_K780002), the open reading frame of the Gastrodia anti-fungal protein 1 (GAFP-1) and a double terminator BBa_B0015. optimized for its expression and its secretion in Bacillus subtilis. The codon optimization was made thanks to the DNA 2.0 software program.

Type

Composite part

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373

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This expression cassette is designed for the expression and secretion of the Gastrodia anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.

Design

This part is composed of the strong, constitutive promoter of Bacillus subtilis P_veg (BBa_K823003), strong RBS (BBa_K780002), the open reading frame of the Gastrodia anti-fungal protein 1 (GAFP-1) and a double terminator (B0015). optimized for its expression and its secretion in B. subtilis. The codon optimization was made thanks to the DNA 2.0 software program.

Type

Generator

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373

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D4E1 is a linear synthetic peptide of 17 amino acids which has shown to have antifungal activities by complexing with a sterol present in conodial wall of a varety of fungi.

Design

This part is composed of a Strong RBS (BBa_K780002), the open reading frame of D4E1 and a Double terminator (BBa_B0015) optimized for its expression and its secretion in Bacillus subtilis. This part was optimized for the expression and its secretion in B. subtilis thanks to the DNA 2.0 software program.

Type

Translational unit

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.
- Can J Microbiol. 1998 Jun;44(6):514-20.

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D4E1 is a linear synthetic peptide of 17 amino acids which has shown to have antifungal activities by complexing with a sterol present in conodial wall of a varety of fungi.

Design

This part is composed of the strong, constitutive promoter of Bacillus subtilis P_veg (BBa_K823003), a strong RBS (BBa_K780002), the open reading frame of D4E1 and a double terminator (BBa_B0015). This part was optimized for the expression and its secretion in B.subtilis thanks to the DNA 2.0 software program.

Type

Translational unit

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.
- Can J Microbiol. 1998 Jun;44(6):514-20.

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EcAMP-1 is an antimicrobial peptide of 37 amino acids originated from the specie Echinochloa crus-galli, a type of wild grass. This peptide has a particular structure : it is helical because of two disulfide bonds.
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.
This part was added to the Registry by the iGEM Utah State team in 2013.

Design

This part is composed of the constitutive promoter P_veg and strong RBS for Bacillus subtilis (BBa_K733013) and the open reading frame of EcAMP-1.
The EcAMP-1 part was codon optimized for E. coli by the iGEM Utah State team and thanks to the Life Technologies GeneArt software program.

Type

Generator

Tests

This part is not tested yet.

References

Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli). The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153

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EcAMP-1 is an antimicrobial peptide of 37 amino acids originated from the specie Echinochloa crus-galli, a type of wild grass. This peptide has a particular structure : it is helical because of two disulfide bonds.
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.
This part was added to the Registry by the iGEM Utah State team in 2013.

Design

This part is composed of the constitutive promoter P_veg and strong RBS for Bacillus subtilis (BBa_K733013), the open reading frame of EcAMP-1 and a double terminator (BBa_B0015).
The EcAMP-1 part was codon optimized for Escherichia coli by the iGEM Utah State team and thanks to the Life Technologies GeneArt software program.

Type

Generator

Tests

This part is not tested yet.

References

Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli). The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153

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EcAMP-1 is an antimicrobial peptide of 37 amino acids originated from the specie Echinochloa crus-galli, a type of wild grass. This peptide has a particular structure : it is helical because of two disulfide bonds.
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.
This part was added to the Registry by the iGEM Utah State team in 2013.
We observed the presence of the stop codon in the suffix. When we have digested this Biobrick, it disappeared. So we added this codon in upstream of the suffix and now, we can reuse this Biobrick.

Design

This part is composed of the constitutive promoter P_veg and strong RBS for Bacillus subtilis (BBa_K733013), the revised open reading frame of EcAMP-1 (with a stop codon) and a double terminator (BBa_B0015).
The EcAMP-1 part was codon optimized for Escherichia coli by the iGEM Utah State team and thanks to the Life Technologies GeneArt software program.

Type

Composite

Tests

This part was not tested yet

References

Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli). The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153

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Design

This part is designed for the co-expression of two different peptides with anti-fungal activities : D4E1 and GAFP-1. It is composed of the strong, constitutive promoter of Bacillus subtilis P_veg (BBa_K823003) and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).

Type

Generator

Tests

This part was tested on the fungi Trichoderma reesei (See Fungicides module)

References

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This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter P_veg. This construction has been checked by sequencing and has shown to work also in Escherichia coli

Design

This part is composed of a constitutive promoter P_veg (BBa_K823003), spoVG RBS (BBa_KK143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_KB0015)

Type

Reporter

Tests

This part was tested in both E. coli and B. subtilis. The sequences was verified by sequencing.

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This part is designed to enable the expression of a Red Fluorescent Protein in Bacillus subtilis under the control of a constitutive promoter P_lepA. This construction has been checked by sequencing and has shown to work also in Escherichia coli

Design

This part is composed of a constitutive promoter P_lepA (BBa_K823002), spoVG RBS (BBa_K143021), the coding sequence of the RFP (E1010 from BBa_K606013) and a double terminator (BBa_B0015)

Type

Reporter

Tests

This part was tested in both E. coli and B. subtilis. The sequences was verified by sequencing.

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P_lepA is a constitutive promoter in Bacillus subtilis (BBa_K823002) coupled with a RBS spoVG (BBa_K143021). To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency. This construction is also working with Escherichia coli and has been verified by sequencing.

Type

Composite

Tests

This part was checked by sequencing

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This part is an empty backbone vector for the usage in Bacillus subtilis. It is on biobrick version of pKL190 which integrates in the thrC locus and can be selected with Erythromycin . It has an Ampicillin resistance for cloning in Escherichia coli. The backbone contains an RFP in the BioBrick site (BBa_J04450) to facilitate the cloning in E.coli.
The handling of this type of vector is described here.

Design

We digested pKL190 plasmid and PCR products of BBa_J04450 with BamHI and EcoRI. We proceded to ligation.

Type

Backbone

Tests

This part was tested by transforming B. subtilis. Clones obtained were Threonine-dependant and resistant to Erythromycin (10µg/ml).

References

R. Bernard, K.A. Marquis, and D.Z. Rudner. Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilisMol Microbiol. Nov 2010; 78(4): 866–882.

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