IGEM:IMPERIAL/2008/Prototype/Wetlab/parts

• James Chappell: Just realised I cannot submit the promoters to the registry because unfortunately my account is for 2007. Ill update it later, but for now ill load to this page.

• Chris D Hirst 21:40, 27 August 2008 (EDT):Had a go at uploading a sequence to the registry last night with mixed results, will check on this later and improve...

• Chris D Hirst 04:02, 28 August 2008 (EDT):Did some more work, Part:K143001 is now up on the parts registry, I'l put it's sister part (part:BBa_K143002) up asap. Before we upload anymore however I think we should write out ALL the basic parts and asign them codes to prevent problems downstream. Linking via the bbpart system (see iGEM teams page on OWW) doesn't actually work....

Tom (or James) would it be possible for one of you to make a nice diagram (or two interlinking ones - 1 for 5', 1 for 3') to signify integration sequences?

• Chris D Hirst 07:36, 28 August 2008 (EDT):Ok, the <bbpart></bbpart> system does work, but only on registry pages....

Integration pic

Here is the picture for integration, I can alter it if you want - James

Numbering/Coding Rules!

• Chris D Hirst 07:53, 28 August 2008 (EDT):All Imperial iGEM 2008 parts are currently being placed in our allocated K413000-K413999 space; the divisions our below, so when adding a part try to ensure it is placed in the correct section. Also, I've place a list of codes at the bottom of this page and assigned all our basic parts and some potential parts codes (though they can be changed - just let me know)

Basic Parts:

K413000-K413009 Integration sequences

K413010-K413019 Promoters

K413020-K413029 RBS

K413030-K413039 Coding Regions (Complete Genes - excludes those with coding sequences, they are separate)

K413040-K413049 Tags (ie. Secretion signals) and Tagged coding regions

Composite Parts:

K413050-K413089

Composite Parts Page

Construction intermediates?:

K413090-K413100

Adding Parts to the Registry

The registry has a simple guide about adding parts on the following link. Before we start to add our parts we should collect the following information about each of our parts:

• A part name
• The DNA sequence of the part you are making
• A short description of the part
• A long description of the part, including references
• The source of the part, including references
• Design Considerations

Most of this information is on the wiki under the Sequence Page. If the wet lab team could all contribute to adding these parts it would help speed things up. Once we have our parts we can then build up our constructs that we will be submitting to the registry.

Promoters

Promoter ctc

• Part name = promoter ctc
• Sequence =
• Promoter ctc is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress from nutrient and physical stress response. The context with which we used the promoter ctc, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output.

• Promoter ctc is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol[1]. The promoter ctc has been used previously as a read out for the activation of sigma factor B [2].
• The context with which we used the promoter ctc, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in B.subtilis [3].

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [2][3]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter ctc sequence.

References

1. Zhang S and Haldenwang WG. Contributions of ATP, GTP, and redox state to nutritional stress activation of the Bacillus subtilis sigmaB transcription factor. J Bacteriol. 2005 Nov;187(22):7554-60. DOI:10.1128/JB.187.22.7554-7560.2005 |
2. Igo MM and Losick R. Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol. 1986 Oct 20;191(4):615-24. DOI:10.1016/0022-2836(86)90449-3 |
3. Suzuki N, Takaya N, Hoshino T, and Nakamura A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J Gen Appl Microbiol. 2007 Apr;53(2):81-8. DOI:10.2323/jgam.53.81 |

Promoter hyper-spank

• Part name = promoter hyper-spank
• Sequence =
• Promoter hyper-spank is an inducible promoter that has been designed for high expression in B.subtilis. Gene expression under the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The context with which we used the promoter hyper-spank, was to take an input of IPTG and give Polymerase Per Second(PoPS) as an output.

• Promoter hyper-spank is an inducible promoter that has been designed for high expression in B.subtilis. Gene expression under the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The context with which we used the promoter hyper-spank, was to take an input of IPTG and give Polymerase Per Second(PoPS) as an output. IPTG does not induce the promoter hyper-spank directly, but requires the transcriptional regulator LacI, (<bbpart>BBa_K413035</bbpart>). This means that LacI must be constitutively expressed in B.subtilis in order to use the promoter hyper-spank.

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [1][2][3]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter hyper-spank sequence.

References

1. Geng H, Nakano S, and Nakano MM. Transcriptional activation by Bacillus subtilis ResD: tandem binding to target elements and phosphorylation-dependent and -independent transcriptional activation. J Bacteriol. 2004 Apr;186(7):2028-37. DOI:10.1128/JB.186.7.2028-2037.2004 |
2. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol. 2002 Sep;184(17):4881-90. DOI:10.1128/JB.184.17.4881-4890.2002 |
3. Ferguson CC, Camp AH, and Losick R. gerT, a newly discovered germination gene under the control of the sporulation transcription factor sigmaK in Bacillus subtilis. J Bacteriol. 2007 Nov;189(21):7681-9. DOI:10.1128/JB.01053-07 |

Promoter xylose

• Part name = promoter xylose
• Sequence =
• Promoter Xylose is an inducible promoter that has been designed for high expression in B.subtilis. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give Polymerase Per Second(PoPS) as an output.

• Promoter xylose is an inducible promoter that has been designed for high expression in B.subtilis. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give Polymerase Per Second(PoPS) as an output. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator XylR, (<bbpart>BBa_K413036</bbpart>) This means that XylR must be constitutively expressed in B.subtilis in order to use the promoter xylose.

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [1][2]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter xylose sequence.

References

1. Kim L, Mogk A, and Schumann W. A xylose-inducible Bacillus subtilis integration vector and its application. Gene. 1996 Nov 28;181(1-2):71-6. DOI:10.1016/s0378-1119(96)00466-0 |
2. Härtl B, Wehrl W, Wiegert T, Homuth G, and Schumann W. Development of a new integration site within the Bacillus subtilis chromosome and construction of compatible expression cassettes. J Bacteriol. 2001 Apr;183(8):2696-9. DOI:10.1128/JB.183.8.2696-2699.2001 |

Promoter gsiB

• Part name = promoter gsiB
• Sequence =
• Promoter gsiB is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress from nutrient and physical stress response. The context with which we used the promoter gsiB, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output.

• Promoter gsiB is a sigma factor B dependent promoter found in B.subtilis. In B.subtilis endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol[1]. The promoter gsiB has been used previously as a read out for the activation of sigma factor B [2].
• The context with which we used the promoter gsiB, was to take blue light as an input and give Polymerase Per Second(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in B.subtilis [3].

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [2][3]. This sequence was then synthesised by Geneart.

• Design - Biobrick standard was applied to the promoter gsiB sequence.

References

1. Zhang S and Haldenwang WG. Contributions of ATP, GTP, and redox state to nutritional stress activation of the Bacillus subtilis sigmaB transcription factor. J Bacteriol. 2005 Nov;187(22):7554-60. DOI:10.1128/JB.187.22.7554-7560.2005 |
2. Nguyen HD, Nguyen QA, Ferreira RC, Ferreira LC, Tran LT, and Schumann W. Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid. 2005 Nov;54(3):241-8. DOI:10.1016/j.plasmid.2005.05.001 |
3. Suzuki N, Takaya N, Hoshino T, and Nakamura A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J Gen Appl Microbiol. 2007 Apr;53(2):81-8. DOI:10.2323/jgam.53.81 |

Promoter 43

• Part name = promoter P43
• Sequence =
• Promoter P43 is constitutive promoter found in B.subtilis. The context with which we used the promoter P43 is as a Polymerase Per Second (PoPS) generator.

• Promoter 43 is a constitutive promoter that constitutively expresses the P43 protein in B.subtilis. This promoter has been shown to be recognized and active during the exponential and lag phases of growth. It has been hypothesized that the ability to recognize the promoter in exponential and lag phase of growth is due to the recognition of the promoter by both sigma factor 55 (the major sigma factor) and sigma factor 37 (the lag phase sigma factor)[1]. The P43 promoter has been previously used for constitutive expression of exogenous genes within B.subtilis vectors[2].
• The context with which we used the promoter P43 is as a Polymerase Per Second (PoPS) generator.

• Source - The part was designed using the sequence from the B.subtilis genome and from previously published papers [2]. This sequence was then synthesised by Geneart.

• Design - The biobrick part was designed to include the binding sites for both the sigma factor 55 and 37. In addition the biobrick standard was applied to the promoter P43 sequence.

References

1. Zhang XZ, Cui ZL, Hong Q, and Li SP. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl Environ Microbiol. 2005 Jul;71(7):4101-3. DOI:10.1128/AEM.71.7.4101-4103.2005 |
2. Wang PZ and Doi RH. Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem. 1984 Jul 10;259(13):8619-25.

Promoter Pveg

• Part name = promoter Pveg
• Sequence =
• Pveg constitutive promoter for B.subtilis.

• Pveg is a constitutive promoter that constitutively expresses the P43 protein in B.subtilis. Pveg contains binding sites for the B.sutbilis major sigma factor[1]. Pveg in B.subtilis utilises two binding sites to cause high expression of genes[2], however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how[3].

• The context with which we used the promoter Pveg is as a Polymerase Per Second (PoPS) generator.

• Source - The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database[3] then a section containing the binding site synthesised by Geneart.

• Design - The biobrick part was designed to include a single binding site for the B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.

References

1. Moran CP Jr, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, and Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339-46. DOI:10.1007/BF00729452 |
2. Le Grice SF and Sonenshein AL. Interaction of Bacillus subtilis RNA polymerase with a chromosomal promoter. J Mol Biol. 1982 Dec 15;162(3):551-64. DOI:10.1016/0022-2836(82)90388-6 |
3. Molle V, Fujita M, Jensen ST, Eichenberger P, González-Pastor JE, Liu JS, and Losick R. The Spo0A regulon of Bacillus subtilis. Mol Microbiol. 2003 Dec;50(5):1683-701. DOI:10.1046/j.1365-2958.2003.03818.x |
4. Sierro N, Makita Y, de Hoon M, and Nakai K. DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res. 2008 Jan;36(Database issue):D93-6. DOI:10.1093/nar/gkm910 |

RBS

GsiB

Name:GsiB

Sequence:

Description: GsiB is an endogenous ribosome binding site from B.subtilis. The sequence of the gsiB ribosome binding site is AAAGGAGG which is complementary to the sequence UUUCCUCC from the 3' region of the 16s rRNA from B.subtilis.

GsiB is an endogenous ribosome binding site (RBS) from B.subtilis. The sequence of the gsiB ribosome binding site is AAAGGAGG which is complementary to the sequence UUUCCUCC from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -9.3kcal.

Source:The sequence was taken from a previous research paper [1] and was constructed by Geneart.

Design:In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.

1. Jürgen B, Schweder T, and Hecker M. The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site. Mol Gen Genet. 1998 Jun;258(5):538-45. DOI:10.1007/s004380050765 |
2. Moran CP Jr, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, and Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339-46. DOI:10.1007/BF00729452 |

SpoVG

Name:SpoVG

Sequence:

Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal.

Source:The sequence was taken from a previous research paper [1] and was constructed by Geneart.

Design:In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.

1. Jürgen B, Schweder T, and Hecker M. The stability of mRNA from the gsiB gene of Bacillus subtilis is dependent on the presence of a strong ribosome binding site. Mol Gen Genet. 1998 Jun;258(5):538-45. DOI:10.1007/s004380050765 |
2. Moran CP Jr, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, and Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339-46. DOI:10.1007/BF00729452 |

Integration Sequences

AmyE

5'

Name: 5’ AmyE Integration Sequence

Code: BBa_K143001

Sequence:

Short: 5’ integration sequence for the AmyE locus of B.subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome.The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

The AmyE locus was the first locus used for integration into B.subtilis by Shimotsu and Henner[1] and is still commonly used in vectors such as pDR111[2], pDL[3] and their derivatives. Integration at the AmyE locus removes the ability of B.subtilis to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn[4]. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome.

Source: The 5’ integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on B.subtilis, in particular in the studies of transcriptional control[2, 5, 6]

Design: The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration

References

1. Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene. 1986;43(1-2):85-94. DOI:10.1016/0378-1119(86)90011-9 |
2. Nakano S, Küster-Schöck E, Grossman AD, and Zuber P. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13603-8. DOI:10.1073/pnas.2235180100 |

3. Bacillus Genetic Stock Center [www.bgsc.org]

4. Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27–74.

5. Erwin KN, Nakano S, and Zuber P. Sulfate-dependent repression of genes that function in organosulfur metabolism in Bacillus subtilis requires Spx. J Bacteriol. 2005 Jun;187(12):4042-9. DOI:10.1128/JB.187.12.4042-4049.2005 |
6. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol. 2002 Sep;184(17):4881-90. DOI:10.1128/JB.184.17.4881-4890.2002 |

3'

Name: 3’ AmyE Integration Sequence

Code: BBa_K143002

Sequence:

Short: 3’ integration sequence for the AmyE locus of B.subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143001) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

The AmyE locus was the first locus used for integration into B.subtilis by Shimotsu and Henner[1] and is still commonly used in vectors such as pDR111[2], pDL[3] and their derivatives. Integration at the AmyE locus removes the ability of B.subtilis to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn[4]. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome.

Source: The 3’ integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on B.subtilis, in particular in the studies of transcriptional control[2, 5, 6]

Design: The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 3' end of the gene is the 3' sequence required for integration

References

1. Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene. 1986;43(1-2):85-94. DOI:10.1016/0378-1119(86)90011-9 |
2. Nakano S, Küster-Schöck E, Grossman AD, and Zuber P. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13603-8. DOI:10.1073/pnas.2235180100 |

3. Bacillus Genetic Stock Center [www.bgsc.org]

4. Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27–74.

5. Erwin KN, Nakano S, and Zuber P. Sulfate-dependent repression of genes that function in organosulfur metabolism in Bacillus subtilis requires Spx. J Bacteriol. 2005 Jun;187(12):4042-9. DOI:10.1128/JB.187.12.4042-4049.2005 |
6. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol. 2002 Sep;184(17):4881-90. DOI:10.1128/JB.184.17.4881-4890.2002 |

EpsE

5'

Name: 5’ EpsE Integration Sequence

Code: BBa_K143005

Sequence:

Short: 5’ Integration Sequence for the EpsE locus of B.subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome.The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143006) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

The EpsE (aka YveO) locus has to our knowledge never been used for integration into B.subtilis before, but is useful in that it knocks out the potential molecular clutch EpsE gene [1]. In particular, both the 5' and 3' integration sequences for the EpsE locus conatin in-frame stop codons to prevent translation of the gene (if nothing is integrated into the locus, integration also prevents correct EpsE expression). The 5' and 3' integration sequences for the EpsE locus were used to integrate over the EpsE gene and prevent its expression in the Imperial 2008 iGEM project B.sutbilis host.

Source: The 5’ integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosme from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. It was produced by PCR cloning with Pfu

Design: The EpsE integration sequences were designed from the EpsE (aka YveO) gene's Genbank entry[2] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu

References

1. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008 Jun 20;320(5883):1636-8. DOI:10.1126/science.1157877 |

2. http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene, Gene ID:938633

3'

Name: 3’ EpsE Integration Sequence

Code: BBa_K143006

Sequence:

Short: 3’ Integration Sequence for the EpsE locus of B.subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143005) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

The EpsE (aka YveO) locus has to our knowledge never been used for integration into B.subtilis before, but is useful in that it knocks out the potential molecular clutch EpsE gene [1]. In particular, both the 5' and 3' integration sequences for the EpsE locus conatin in-frame stop codons to prevent translation of the gene (if nothing is integrated into the locus, integration also prevents correct EpsE expression). The 5' and 3' integration sequences for the EpsE locus were used to integrate over the EpsE gene and prevent its expression in the Imperial 2008 iGEM project B.sutbilis host.

Source: The 3’ integration sequence was taken from the B.subtilis chromosome and is homologous to the middle section of the EpsE gene. It was produced by PCR cloning with Pfu

Design: The EpsE integration sequences were designed from the EpsE (aka YveO) gene's Genbank entry[2] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu

References

1. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008 Jun 20;320(5883):1636-8. DOI:10.1126/science.1157877 |

2. http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene, Gene ID:938633

PyrD

5'

Name: 5’ PyrD Integration Sequence

Code: BBa_K143003

Sequence:

Short: 5’ Integration Sequence for the PyrD locus of B. subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (<bbpart> BBa_K143004</bbpart>) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) [1].

Integration into the PyrD locus makes the B.subtilis auxotrophs for uracil and transformants require about 40μg/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negativly select for transformants.

Source:

Design:

3'

Name: 3’ PyrD Integration Sequence

Code: BBa_K143004

Sequence:

Short: 3’ Integration Sequence for the PyrD locus of B. subtilis

Long: Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (<bbpart> BBa_K143003</bbpart>) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis.

Source:

Design:

Coding Regions

YtvA

Name:YtvA

Code:

Descriptions: YtvA is a blue light receptor endogenously found in B.subtilis. Activation of the YtvA light receptor activates the mild stress response pathway, accumulating in sigma B activation. YtvA can be over expressed to enhance the sigma B output to the blue light input.

Description-longer:YtvA is a blue light receptor endogenously found in B.subtilis. Activation of the YtvA light receptor activates the mild stress response pathway, accumulating in sigma B activation. Previous research has shown that sigma B dependent transcription in response to blue light light exposure can be enhanced by the over expression of the YtvA receptor [1]. We over expressed YtvA to enhance the transcriptional output of sigma B promoters in response to blue light.

References

1. Suzuki N, Takaya N, Hoshino T, and Nakamura A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J Gen Appl Microbiol. 2007 Apr;53(2):81-8. DOI:10.2323/jgam.53.81 |

EspE

Name: EpsE

Code: BBa_K143032

Sequence:

Short: EpsE Molecular Clutch Gene

Long: The epsE gene of the exopolysaccharide synthesis operon of B. subtilis has been suggested to function in a manor similar to a molecular clutch[1]. If expressed inside a cell it will prevent flagellar movement causing the cell to no longer be able to swim effectively and instead only tumble. As such EpsE could potentially be used as a controller of B. subtilis movement.

Though the EPS operon is normally repressed in B. subtilis, if EpsE is synthetically expressed it would be beneficial for the original copy of epsE to be knocked out. This can be achieved by integrating over the EesE gene with the epsE integration Biobricks (<bbpart>BBa_K143005</bbpart> and <bbpart>BBa_K143006</bbpart>) which contain 2 in-frame stop codons.

Although many bacterial flaggelar assemblies contain proteins that are similar in shape, there is no guarantee that the epsE gene will function correctly in any host cell other than B. subtilis

Source: The epsE gene sequence was taken from the B. subtilis chromosome and was synthesised by GeneArt.

Design: The epsE(aka yveO) sequence was located in the B. subtilis chromosome[1] and the PstI restriction site removed before synthesis by GeneArt

References

1. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessières P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Cordani JJ, Connerton IF, Cummings NJ, Daniel RA, Denziot F, Devine KM, Düsterhöft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Hènaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauël C, Médigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O'Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Togoni A, Tosato V, Uchiyama S, Vandebol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, and Danchin A. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997 Nov 20;390(6657):249-56. DOI:10.1038/36786 |
1. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008 Jun 20;320(5883):1636-8. DOI:10.1126/science.1157877 |

Aad9

Name: Aad9

Code: BBa_K143031

Sequence:

Short: Aad9 Spectinomycin Resistance Gene

Long: Aad9 is the spectinomycin resistance gene from Enterococcus faecalis[1]. Expression in a host confers resistance to spectinomycin at a concentration of 100μg/μl and has been used in a variety of vectors for both B. subtilis and E. coli including pDP870[2], pCOMT-Kan[3] and pIEF16s[4]

Source: Aad9 was PCR cloned from the B. subtilis integration vector pDR111 using Pfu DNA polymerase

Design: The sequence of B. subtilis integration vector pDR111 was searched for the spectinomycin resistance gene and the Biobrick standard applied to the gene sequence

References

1. LeBlanc DJ, Lee LN, and Inamine JM. Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis. Antimicrob Agents Chemother. 1991 Sep;35(9):1804-10. DOI:10.1128/AAC.35.9.1804 |
2. Klijn A, Moine D, Delley M, Mercenier A, Arigoni F, and Pridmore RD. Construction of a reporter vector for the analysis of Bifidobacterium longum promoters. Appl Environ Microbiol. 2006 Nov;72(11):7401-5. DOI:10.1128/AEM.01611-06 |
3. Staddon JH, Bryan EM, Manias DA, and Dunny GM. Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria. J Bacteriol. 2004 Apr;186(8):2393-401. DOI:10.1128/JB.186.8.2393-2401.2004 |
4. Zhang XZ, Yan X, Cui ZL, Hong Q, and Li SP. mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res. 2006 May 19;34(9):e71. DOI:10.1093/nar/gkl358 |

XylR

Name: XylR

Code: BBa_K143036

Sequence:

Short: Xylose operon regulatory protein

Long: Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as xylose.

XylR if the regulator protein for the Xylose operon in B. subtilis[1] and is responsible for ensuring that in the absence of xylose the xylose metabolism proteins are not expressed. Though endogenous to B. subtilis, to minimise the leakage of a xylose inducible promoter XylR should be over-expressed. In the presence of xylose, the XylR tetramer is unable to bind DNA and so transcription resumes.

It must be noted that in all B. subtilis strains that do not have the Xylose operon knocked out the xylose inducer will gradually be metabolised by the host

XylR was used in conjunction with the Xylose operon promoter (<bbpart>BBa_K143014<bbpart>) and acted as an input adaptor for a Polymerases per second (POPS) output

Source: The XylR protein was PCR cloned form the B. subtilis genome using Pfu DNA polymerase

Design: The XylR protein was identified in the genome using its Genbank entry[2] and NCBI's sequence viewer and PCR primers designed from the sequence. Biobrick prefix and suffix sequences were added and the gene cloned by PCR with Pfu DNA polymerase

References

1. Kreuzer P, Gärtner D, Allmansberger R, and Hillen W. Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol. 1989 Jul;171(7):3840-5. DOI:10.1128/jb.171.7.3840-3845.1989 |

2. http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene, Gene ID:939531

LacI (N-terminal deletion, Lva^-)

Name: LacI

Code: BBa_K143033

Sequence:

Short: LacI (Lva^-, N-terminal deletion) regulatory protein

Long: LacI is a regulatory protein responsible for the repression of many catabolite genes. Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as IPTG or certain sugars.

LacI if the regulator protein for the lactose operon in E.coli and the hyper-spank protein of B. subtilis[1](<bbpart>BBaK143015</bbpart>) and is responsible for ensuring that in the absence of lactose (or IPTG) that there is no expression trough these promoter. LacI is not endogenous to B. subtilis, so LacI will need to be expressed in the host in order for the hyper-spank promoter to be regulated. In the presence of IPTG or lactose, the LacI tetramer is unable to bind DNA and so transcription resumes.

This version of LacI lacks a Lva degradation tag and has a small(3 amino acid) N-terminal deletion relative to the current registry LacI (<bbpart>BBa_C0012</bbpart)> and is derivatives. The N-terminal deletion appears to be common to most of the LacI genes used in conjunction with B. subtilis though both forms are found in E.coli (in differing strains).

LacI was used in conjunction with the Hyper-spank promoter (<bbpart>BBa_K143015<bbpart>) and acted as an input adaptor for a Polymerases per second (POPS) output

Source: The LacI gene was cloned fromB. subtilis shuttle vector pDR111 using Pfu DNA polymerase PCR

Design: LacI was located in the sequence of the B. subtilis shuttle vector pDR111. This version of LacI lacks a Ltva degradation sequence and has a small N-terminal deletion that is observed in many LacI used in studies on B.subtilis. In particular, this LacI protein is used in pDR111 to regulate expression of the inducible Phyper-spank protein (<bbpart>BBa_K143015</bbpart>) (also used in the pDR111 vector). The Biobrick prefix and suffix were applied to the gene

References

1. Silvaggi JM, Perkins JB, and Losick R. Small untranslated RNA antitoxin in Bacillus subtilis. J Bacteriol. 2005 Oct;187(19):6641-50. DOI:10.1128/JB.187.19.6641-6650.2005 |

Biomaterials & Signal Peptides

EAK16-II & LipA

Name: LipA-EAK16II Fusion Protein

Code: BBa_K413034

Sequence:

Description: EAK16-II is a sixteen amino acid peptide that self-assembles to form β-sheet structures in an aqueous medium. The alternating positive and negative charges (--++--++) are responsible for creating an electrostatic attraction between adjacent peptides [1], triggering self-assembly when the EAK16-II peptides are exposed to physiological media or salt solution. When examined under SEM, a well-ordered nanofibre structure is formed by the association of the EAK16-II peptides and these nanofibres can futher aggregate to form a membranous 3D scaffold.

LipA is a signal peptide from the B.subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[2]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis.

Source: EAK16-II was identified as a region in zuotin, a Z-DNA binding protein from the yeast genome. LipA originated from the B. subtilis genome[3]. Both components were produced as a fusion protein by GeneArt.

Design: BioBrick standard was applied to LipA-EAK16II Fusion Protein.

Reference:

1. Zhang S, Gelain F, and Zhao X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol. 2005 Oct;15(5):413-20. DOI:10.1016/j.semcancer.2005.05.007 |
2. Ling Lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, and Chun Xia Hu. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv. 2007 Jan-Feb;25(1):1-12. DOI:10.1016/j.biotechadv.2006.08.002 |
3. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, and van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev. 2000 Sep;64(3):515-47. DOI:10.1128/MMBR.64.3.515-547.2000 |

Human Elastin Polypeptide & LipA

Name: LipA-Human Elastin(EP20-24-24) Fusion Protein

Code: BBa_K413035

Sequence:

Description: Elastin is a polymeric extracellular matrix protein found in tissues that require the ability to extend and recoil. Examples of elastin containing tissues include arteries, lungs, ligaments and skin.

Construct EP20-24-24 for human elastin polypeptide consists of distinct exons which code for alternating hydrophobic regions and crosslinking domains from the human elastin polypeptide gene [1].

Under appropriate conditions of temperature and ionic strength, elastin polypeptide undergoes a self-aggregation process known as coacervation. Coacervation is usually induced by an increase in temperature and causes the protein to separate from the solution as a second phase. Unlike most proteins which undergo denaturation when the temperature of the solution increases, elastin polypeptides become more ordered through coacervation [2].

LipA is a signal peptide from the B.subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis.

Source: All exons in EP20-24-24 are derived from the human elastin polypeptide gene. EP20-24-24 was used to study the effect of various combinations of exons on coacervation of elastin polypeptide. LipA originated from the B. subtilis genome[4]. Both components were produced as a fusion protein by GeneArt.

Design: BioBrick standard was applied to the LipA-Human Elastin(EP20-24-24) Fusion Protein.

Reference:

1. Bellingham CM, Woodhouse KA, Robson P, Rothstein SJ, and Keeley FW. Self-aggregation characteristics of recombinantly expressed human elastin polypeptides. Biochim Biophys Acta. 2001 Nov 26;1550(1):6-19. DOI:10.1016/s0167-4838(01)00262-x |
2. Keeley FW, Bellingham CM, and Woodhouse KA. Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin. Philos Trans R Soc Lond B Biol Sci. 2002 Feb 28;357(1418):185-9. DOI:10.1098/rstb.2001.1027 |
3. Ling Lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, and Chun Xia Hu. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv. 2007 Jan-Feb;25(1):1-12. DOI:10.1016/j.biotechadv.2006.08.002 |
4. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, and van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev. 2000 Sep;64(3):515-47. DOI:10.1128/MMBR.64.3.515-547.2000 |

EAK16-II & SacB

Name: SacB-EAK16-II Fusion Protein

Code: BBa_K413038

Sequence:

Description: EAK16-II is a sixteen amino acid peptide that self-assembles to form β-sheet structures in an aqueous medium. The alternating positive and negative charges (--++--++) are responsible for creating an electrostatic attraction between adjacent peptides [1], triggering self-assembly when the EAK16-II peptides are exposed to physiological media or salt solution. When examined under SEM, a well-ordered nanofibre structure is formed by the association of the EAK16-II peptides and these nanofibres can futher aggregate to form a membranous 3D scaffold.

SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium. SacB has been successfully used in the secretion of heterologous proteins such as acid-stable α-amylase, cystatin and interleukin-3 by B.subtilis [2].

Source: EAK16-II is a segment from zuotin, a yeast protein. SacB was identified from the initial part of certain preprotein genes that utilises the the Sec-SRP secretory pathway [3]. Both components were synthesised as a fusin protein by GeneArt.

Design: BioBrick standard was applied to the SacB-EAK16-II Fusion Protein.

Reference:

1. Zhang S, Gelain F, and Zhao X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol. 2005 Oct;15(5):413-20. DOI:10.1016/j.semcancer.2005.05.007 |
2. Ling Lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, and Chun Xia Hu. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv. 2007 Jan-Feb;25(1):1-12. DOI:10.1016/j.biotechadv.2006.08.002 |
3. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, and van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev. 2000 Sep;64(3):515-47. DOI:10.1128/MMBR.64.3.515-547.2000 |

Human Elastin Peptide & SacB

Name: SacB-Human Elastin(EP20-24-24) Fusion Protein

Code: BBa_K413039

Description: Elastin is a polymeric extracellular matrix protein found in tissues that require the ability to extend and recoil. Examples of elastin containing tissues include arteries, lungs, ligaments and skin.

Construct EP20-24-24 for human elastin polypeptide consists of distinct exons which code for alternating hydrophobic regions and crosslinking domains from the human elastin polypeptide gene [1].

Under appropriate conditions of temperature and ionic strength, elastin polypeptide undergoes a self-aggregation process known as coacervation. Coacervation is usually induced by an increase in temperature and causes the protein to separate from the solution as a second phase. Unlike most proteins which undergo denaturation when the temperature of the solution increases, elastin polypeptides become more ordered through coacervation [2].

SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium. SacB has been successfully used in the secretion of heterologous proteins such as acid-stable α-amylase, cystatin and interleukin-3 by B.subtilis [3].

Source: All exons in EP20-24-24 are derived from the human elastin polypeptide gene. EP20-24-24 was used to study the effect of various combinations of exons on coacervation of elastin polypeptide. SacB was identified from the initial part of certain preprotein genes that utilises the the Sec-SRP secretory pathway [4]. Both components were synthesised as a fusin protein by GeneArt.

Design: BioBrick standard was applied to the SacB-Human Elastin(EP20-24-24) Fusion Protein.

Reference:

1. Bellingham CM, Woodhouse KA, Robson P, Rothstein SJ, and Keeley FW. Self-aggregation characteristics of recombinantly expressed human elastin polypeptides. Biochim Biophys Acta. 2001 Nov 26;1550(1):6-19. DOI:10.1016/s0167-4838(01)00262-x |
2. Keeley FW, Bellingham CM, and Woodhouse KA. Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin. Philos Trans R Soc Lond B Biol Sci. 2002 Feb 28;357(1418):185-9. DOI:10.1098/rstb.2001.1027 |
3. Ling Lin Fu, Zi Rong Xu, Wei Fen Li, Jiang Bing Shuai, Ping Lu, and Chun Xia Hu. Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv. 2007 Jan-Feb;25(1):1-12. DOI:10.1016/j.biotechadv.2006.08.002 |
4. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, and van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev. 2000 Sep;64(3):515-47. DOI:10.1128/MMBR.64.3.515-547.2000 |

Codes and associated parts

Note

• Aad9 is the Spectinomycin resistance gene
• RI - Resistance Integration Brick, P - Promoter, Pi - chemically inducible promoter, Pl - light inducible promoter, Bs - B.subtilis, PTC - Promoter Testing Construct, Rep - Repressor protein

Code	Part	Code	Part	Code	Part	Code	Part	Code	Part
K143000		K143001	5' AmyE	K143002	3' AmyE	K143003	5' PyrD	K143004	3' PyrD
K143005	5' EpsE	K143006	3'EpsE	K143007		K143008		K143009
K143010		K143011	Promoter P43	K143012	Promoter Pveg	K143013	Promoter Phyper-spank	K143014	Promoter Pxyl
K143015	Promoter Pctc	K143016	Promoter PgsiB	K143017		K143018		K143019
K143020		K143021	gsiB RBS	K143022	spoVG RBS	K143023		K143024
K143025		K143026		K143027		K143028		K143029
K143030		K143031	Aad 9	K143032	EpsE	K143033	LacI	K143034	LipA-EAK16
K143035	LipA-Elastin	K143036	XylR	K143037	YtvA	K143038	SacB-EAK16	K143039	SacB-Elastin
K143040		K143041		K143042		K143043		K143044
K143045		K143046		K143047		K143048		K143049
K143050	P43-gsiB	K143051	P43-spoVG	K143052	Pveg-gsiB	K143053	Pveg-spoVG	K143054	Phyperspank-gsiB
K143055	Phyper-spank-spoVG	K143056	Pxyl-gsiB	K143057	Pxyl-spoVG	K143058	Pctc-gsiB	K143059	Pctc-spoVG
K143060	PgsiB-gsiB	K143061	PgsiB-spoVG	K143062	CAT - Terminator	K143063	Aad9 - Terminator	K143064	LacI - Terminator
K143065	XylR - Terminator	K143066	Int Open	K143067	RI Brick	K143068	RI-Rep-Pi Brick	K143069	RI-Ytva-Pl Brick
K143070	Bs-PTC	K143071	Int Close	K143072		K143073		K143074
K143075		K143076		K143077		K143078		K143079
K143080		K143081		K143082		K143083		K143084
K143085		K143086		K143087		K143088		K143089
K143090		K143091		K143092		K143093		K143094
K143095		K143096		K143097		K143098		K143099