Team:Toulouse/Result/parts

From 2014.igem.org

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<p class="title1">Parts</p>
 
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>,  
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>,  
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</p>
</p>
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<p class="title2">1. Submitted parts</p>
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<p class="title1">Submitted parts</p>
<p class="texte">
<p class="texte">
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<p class="title3">Chemotaxis</p>
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<p class="title2">Chemotaxis</p>
<!--K1364000 : RBS + Chimio + terminator-->
<!--K1364000 : RBS + Chimio + terminator-->
-
<p class="texte">
+
 
-
<b>BBa_K1364000:</b> Strong RBS for <i>B. subtilis</i>
+
<p class="title3">
-
(<a href="http://parts.igem.org/Part:BBa_K780001">K780001</a>)
+
<a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p>
-
+ Chemotaxis code
+
<p class="texte">This part is designed to enable a N-acetylglucosamine  
-
+ Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br><br>
+
based chemotaxis in <i>Bacillus subtilis</i>.
-
<b>N-acetylglucosamine based chemotaxis:</b><br>
+
<p class="title4">Design:</p>
-
This part is designed to enable a N-acetylglucosamine  
+
<p class="texte">The encoded protein is a chimera of two proteins:<br>
-
based chemotaxis in <i>Bacillus subtilis</i>.<br><br>
+
-
<b>Design:</b><br>
+
-
The encoded protein is a chimera of two proteins:<br>
+
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br>
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br>
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br>
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br>
-
<br><br>
+
<br>
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal:  
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal:  
these domains correspond to the amino acids 1-37 and 282-610.  
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  
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.<br>
+
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p>
-
<br>
+
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png">
-
<b>Type</b><br>
+
<p class="title4">Type</p>
-
Translation unit<br>
+
<p class="texte">Translation unit</p>
-
<br>
+
<p class="title4">Tests</p>
-
<b>Tests</b><br>
+
<p class="texte">This part is not tested yet.</p>
-
This part was tested with the protocol Plug and pond.
+
<p class="title4">References</p>
-
Link ?<br>
+
<p class="texte">Uniprot:<br>
-
<!--à compléter avec les tests--><br>
+
-
<b>References</b><br>
+
-
Uniprot:<br>
+
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br>
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br>
-
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br>
+
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p>
<!--à compléter-->
<!--à compléter-->
</p>
</p>
-
 
<br>
<br>
<!--K1364004 : Promotor Pveg + Chimio-->
<!--K1364004 : Promotor Pveg + Chimio-->
-
<p class="texte">
+
<p class="title3">
-
<b>BBa_K1364004:</b> Strong, constitutive promotor of <i>B. subtilis</i>
+
<a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i>  
-
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) + RBS, Chemotaxis code and Double terminator (K1364000)<br>
+
<br>
<br>
-
<b>N-acetylglucosamine based chemotaxis:</b><br>
+
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p>
-
This part is designed to enable a N-acetylglucosamine  
+
<p class="title4">Design:</p>
-
based chemotaxis in <i>Bacillus subtilis</i>.<br><br>
+
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide,  
-
<b>Design:</b><br>
+
-
This expression cassette is designed for the expression of an antifungal peptide,  
+
D4E1 and for its secretion in <i>Bacillus subtilis</i>.
D4E1 and for its secretion in <i>Bacillus subtilis</i>.
It is composed of the strong, constitutive promotor of <i>B. subtilis</i>  
It is composed of the strong, constitutive promotor of <i>B. subtilis</i>  
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strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),
the open reading frame of D4E1
the open reading frame of D4E1
-
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>) <br>
+
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p>
-
<br>
+
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png">
-
<b>Type</b><br>
+
<p class="title4">Type</p>
-
Expression cassette<br>
+
<p class="texte">Generator</p>
-
<br>
+
<p class="title4">Tests</p>
-
<b>Tests</b><br>
+
<p class="texte">This part was not tested yet.</p>
-
This part was tested with the protocol Plug and pond.
+
<p class="title4">References</p>
-
Link ?<br>
+
<p class="texte">Uniprot:<br>
-
<!--à compléter avec les tests--><br>
+
-
<b>References</b><br>
+
-
Uniprot:<br>
+
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br>
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br>
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br>
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br>
-
<!--à compléter-->
 
-
</p>
 
-
 
-
<br>
 
-
 
-
<!--K1364006 : Pveg, RBS, Chimio, Terminator, Pveg, RBS, Binding and Terminator-->
 
-
<p class="texte">
 
-
<b>BBa_K1364006:</b>
 
-
P<sub>veg</sub>, RBS, Chemotaxis code, Double terminator(BBa_K1364004)
 
-
+ P<sub>veg</sub>, RBS, Binding, Double terminator(BBa_K1364005).<br>
 
-
<b>Type</b><br>
 
-
Expression cassette<br>
 
-
<br>
 
-
<b>Tests</b><br>
 
-
This part was not tested.
 
-
<!--à compléter avec les tests--><br>
 
-
<b>References</b><br>
 
-
See BBa_K1364004 and BBa_1364005.
 
-
<!--à compléter + lien-->
 
</p>
</p>
<br>
<br>
-
<p class="title3">Binding</p>
+
<p class="title2">Binding</p>
<!--K1364001 : RBS + Binding + Terminator-->
<!--K1364001 : RBS + Binding + Terminator-->
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<p class="title3">Fungicides</p>
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<p class="title2">Fungicides</p>
<p class="title4">GAFP-1</p>
<p class="title4">GAFP-1</p>
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<!--SOUS-TITRE-->
<!--SOUS-TITRE-->
-
<p class="title2">2. Used parts</p>
+
<p class="title1">Used parts</p>
<br>
<br>

Revision as of 09:17, 10 October 2014

           
        logo-igem            

Parts

SubtiTree, a bacterium to save our trees

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

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


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 Pveg (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 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
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 Pveg (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 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
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 Pveg 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 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
Composite part

Tests
The electrophoresis and PCR tests were OK.

References


BBa_K1364011: Constitutive promoter Pveg 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 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
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 Pveg (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 Pveg (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 Pveg 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 Pveg (K316001) + RBS-SpoVG (K143021) : Constitutive promotor Pveg and strong RBS for B. subtilis 2014 Kit plate 1, 7H Fungicides (EcAMP)
K823003 Pveg : strong, constitutive promotor of B. subtilis 2014 Kit plate 1, 20G Fungicides (D4E1 GAFP-1)
Chemotaxis
Binding
K823002 PlepA : 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 pSBBS1C for integration into B. subtilis
It integrates in the amyE locus		 Sent by the Munich iGEM team Fungicides
Chemotaxis
Binding
K823022 Empty backbone vector pSBBS4S for integration intoB. subtilis
It integrates in the thrC locus		 Sent by the Munich iGEM team Fungicides
Chemotaxis
Binding
K823021 Reporter vector pSBBS1C-lacZ for B. subtilis Sent by the Munich iGEM team Fungicides
Chemotaxis
Binding


Experimental results
logo
Achievement

iGEM Toulouse 2014

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