Team:Toulouse/Result/parts/Submitted parts

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<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>,
 
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it was sometimes hard for us to get or find desired parts.<br>
 
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However, in collaboration with other iGEM teams and with a synthesis compagny, we managed to obtain or make the following parts:<br>
 
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</p>
 
<p class="title1">Submitted parts</p>
<p class="title1">Submitted parts</p>

Revision as of 16:41, 15 October 2014

           
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Parts

What did we send to the Registry?

Results   >   Parts

Submitted parts

We have deposed 16 new BioBrick parts to the Registry. All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.

I. Chemotaxis

- BBa_K1364000: N-acetylglucosamine based chemotaxis for Bacillus subtilis

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

Design

The encoded protein is a chimera of two proteins:
- the methyl accepting Chemotaxis protein (McpA from Bacillus 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


- BBa_K1364004: Pveg + N-acetylatedglucosamine based chemotaxis for B. subtilis

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 Bacillus subtilis. It is composed of the strong, constitutive promotor of B. subtilis Pveg (K823003), strong RBS for B. subtilis (K780002), the open reading frame of D4E1 and a double terminator (B0015)

Type

Generator

Tests

This part was not tested yet.

References

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


II. Binding

- BBa_K1364005: Pveg + Chitin Binding Cell Wall protein

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


III. Fungicides

GAFP-1


- BBa_K1364002: RBS - Antifungal GAFP-1

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 (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


- BBa_K1364007: RBS + Antifungal GAFP-1 + Double terminator

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 (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 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


- BBa_K1364008: Pveg - strong RBS - Antifungal GAFP-1 - Double terminator

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 Pveg (K823003), strong RBS (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 Bacillus 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


D4E1


- BBa_K1364003:RBS - Antifungal D4E1 - Double terminator

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 (K780002), the open reading frame of D4E1 and a Double terminator (B0015) optimized for its expression and its secretion in Bacillus subtilis. This part was optimized for the expression and its secretion in Bacillus 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.


- BBa_K1364009:Pveg - RBS - Antifungal D4E1 - Double Terminator

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 promotor of B. subtilis Pveg (K823003), a strong RBS (K780002), the open reading frame of D4E1 and a double terminator (B0015). This part was optimized for the expression and its secretion in Bacillus 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.


EcAMP-1

- BBa_K1364010: Pveg - SpoVG - Antifungal EcAMP-1

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 Pveg and strong RBS for B. subtilis (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


- BBa_K1364011: Pveg - SpoVG - Antifungal EcAMP-1 - Double terminator

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 Pveg and strong RBS for B. subtilis (K733013), the open reading frame of EcAMP-1 and a double terminator (B0015).
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


Fungicide operons

- BBa_K1364013: Pveg - SpoVG - Antifungal EcAMP-1 - Double terminator

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 promotor of B. subtilis Pveg (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

See BBa_K1364002 and BBa_K1364003.


Basic tools

- BBa_K1364015: Pveg + RFP

Design

Coming soon!

Type

Reporter

Tests

This part was not tested yet

References

Coming soon!


- BBa_K1364016: PlepA + RFP

Design

Coming soon!

Type

Reporter

Tests

This part was not tested yet

References

Coming soon!


- BBa_K1364017: PlepA + RBS SpoVG

Design

Coming soon!

Type

Composite

Tests

This part was not tested yet

References

Coming soon!


- BBa_K1364019: Pveg + RBS + Antifungal EcAMP + Double terminator

Design

Coming soon!

Type

Composite

Tests

This part was not tested yet

References

Coming soon!


- BBa_K1364021: Integrative plasmid for Bacillus subtilis

Design

Coming soon!

Type

Composite

Tests

This part was not tested yet

References

Coming soon!


Experimental results
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Achievement

iGEM Toulouse 2014

Retrieved from "http://2014.igem.org/Team:Toulouse/Result/parts/Submitted_parts"