From 2014.igem.org

Parts

As the chassis B. subtilis is not as used as E. coli, it was sometimes hard for us the get or find desired parts.
However, with the collaboration of other iGEM and a synthesis compagny, we managed to obtain or make the following parts:

Submitted parts

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

Chemotaxis

BBa_K1364000: Strong RBS for B. subtilis (K780001) + Chemotaxis code + Double terminator (B0015)

N-acetylglucosamine based chemotaxis:
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 was tested with the protocol Plug and pond. Link ?

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

BBa_K1364004: Strong, constitutive promotor of B. subtilis P_veg (K823003) + RBS, Chemotaxis code and Double terminator (K1364000)

N-acetylglucosamine based chemotaxis:
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 P_veg (K823003), strong RBS for B. subtilis (K780002), the open reading frame of D4E1 and a double terminator (B0015)

Type
Expression cassette

Tests
This part was tested with the protocol Plug and pond. Link ?

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

BBa_K1364006: P_veg, RBS, Chemotaxis code, Double terminator(BBa_K1364004) + P_veg, RBS, Binding, Double terminator(BBa_K1364005).
Type
Expression cassette

Tests
This part was not tested.
References
See BBa_K1364004 and BBa_1364005.

Binding

BBa_K1364001: Consensus RBS for B. subtilis (K090505) + Binding code + Double terminator (B0015)

Binding module:
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
Translation unit

Tests
This part was tested with chitin beeds. Link ?
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

Fungicides

GAFP-1

BBa_K1364002: Strong RBS for B. subtilis (K780002) + GAFP-1

GAFP-1: Gastrodia anti-fungal protein
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which 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
Composite part

Tests
Link ?

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: Strong RBS for B. subtilis (K780002) + GAFP-1 + Double terminator (B0015

GAFP-1: Gastrodia anti-fungal protein
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which 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
Link ?

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: Strong, constitutive promoter of Bacillus subtilis P_veg (K823003 + Strong RBS for B. subtilis (K780002) + GAFP-1 + Double terminator (B0015

GAFP-1: Gastrodia anti-fungal protein
The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which 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 (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
Expression cassette

Tests
Link ?

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: Strong RBS for B. subtilis (K780002) + D4E1 + Double terminator (B0015

D4E1
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
Translation unit

Tests
Link ?

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: Strong, constitutive promotor of B. subtilis P_veg (K823003) + RBS, D4E1 code and Double terminator (K1364003)
D4E1
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 P_veg (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
Expression cassette

Tests
Link ?

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: Constitutive promoter P_veg and strong RBS for B. subtilis (K733013) + EcAMP-1 (K1162001)
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 P_veg 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
Composite part

Tests
The electrophoresis and PCR tests were OK.

References

BBa_K1364011: Constitutive promoter P_veg and strong RBS for B. subtilis (K733013) + EcAMP-1 (K1162001) + Double terminator (B0015)
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 P_veg 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
Expression cassette

Tests
The test does not work yet.

References

Fungicide operons

BBa_K1364012: RBS and GAFP-1 (K1364002) + RBS, D4E1 and double terminator (K1364003)

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 translation unit with D4E1 (BBa_K1364002) and the composite part with GAFP-1 (BBa_K1364003).

Type
Translation unit

Tests

References
See BBa_K1364002 and BBa_K1364003.

BBa_K1364013: Strong, constitutive promotor of B. subtilis P_veg (K823003) + RBS, GAFP-1, RBS, D4E1 and double terminator (K1364012).

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 P_veg (K823003) and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).

Type
Expression cassette

Tests

References
See BBa_K1364002 and BBa_K1364003.

BBa_K1364014: Promotor P_veg RBS spoVG, EcAMP-1 (BBa_K1364010) + RBS, GAFP-1, RBS, D4E1 and double terminator (BBa_K1364012)

Design:
This part contains the three fungicides on the same operon. It is composed of the composite part with EcAMP--1 (BBa_K1364010) and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).

Type
??

Tests

References
See BBa_K1364010, BBa_K1364002 and BBa_K1364003.

Used parts

BioBrick	Description	Origine	Module(s)
K733013	P_veg (K316001) + RBS-SpoVG (K143021) : Constitutive promotor P_veg and strong RBS for B. subtilis	2014 Kit plate 1, 7H	Fungicides (EcAMP)
K823003	P_veg : strong, constitutive promotor of B. subtilis	2014 Kit plate 1, 20G	Fungicides (D4E1 GAFP-1) Chemotaxis Binding
K823002	P_lepA : constitutive promotor of B. subtilis. It is the promotor of the lepA gene of B. subtilis	2014 Kit plate 1, 20E	Fungicides (D4E1) Chemotaxis Binding
K606061	SpoVG RBS for B. subtilis Strong ribosome binding site from subtilis. Works fine in E. coli	2014 Kit plate 1, 15A	Optional
K316027	B. subtilis transformation vector with LacI, targets amyE locus.	2014 Kit plate 1, 2N	Fungicides Chemotaxis Binding
B0015	Double terminator (BBa_B0010 + BBa_B0012)	2014 Kit plate 3, 3F	Fungicides (GAFP-1, EcAMP-1)
K780000	Terminator for B. subtilis	Sent by the Warsaw iGEM team
K780001	Strong RBS for B. subtilis	Sent by the Warsaw iGEM team
K780002	Strong RBS for B. subtilis	Sent by the Warsaw iGEM team
K780003	Strong constitutive promotor for B. subtilis	Sent by the Warsaw iGEM team	Fungicides Chemotaxis Binding
K1162001	EcAMP-1	Sent by the Utah State iGEM team	Fungicides (EcAMP-1)
K823023	Empty backbone vector pSB_BS1C for integration into B. subtilis It integrates in the amyE locus	Sent by the Munich iGEM team	Fungicides Chemotaxis Binding
K823022	Empty backbone vector pSB_BS4S for integration intoB. subtilis It integrates in the thrC locus	Sent by the Munich iGEM team	Fungicides Chemotaxis Binding
K823021	Reporter vector pSB_BS1C-lacZ for B. subtilis	Sent by the Munich iGEM team	Fungicides Chemotaxis Binding

Team:Toulouse/Parts