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 system does work, but only on registry pages....

=Integration pic=

=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: 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.
 * 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

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.

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, ( BBa_K413035 ). 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.

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, ( BBa_K413036 ) 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.

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.

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.

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.

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) 1 pmid=9669336
 * 2) 2 pmid=6181373

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) 1 pmid=9669336
 * 2) 2 pmid=6181373

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) 1 pmid=3019840
 * 2) 2 pmid=14597697
 * 3) 3 Bacillus Genetic Stock Center [www.bgsc.org]
 * 4) 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) 5 pmid=15937167
 * 6) 6 pmid=12169614

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) 1 pmid=3019840
 * 2) 2 pmid=14597697
 * 3) 3 Bacillus Genetic Stock Center [www.bgsc.org]
 * 4) 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) 5 pmid=15937167
 * 6) 6 pmid=12169614

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) 1 pmid=18566286
 * 2) 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) 1 pmid=18566286
 * 2) 2 http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene, Gene ID:938633

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 ( BBa_K143004 ) 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 ( BBa_K143003 ) 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:

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.

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 ( BBa_K143005 and BBa_K143006 ) 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

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

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 ( BBa_K143014 ) 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

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 ( BBaK143015 ) 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 ( BBa_C0012 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 ( BBa_K143015 ) 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 ( BBa_K143015 ) (also used in the pDR111 vector). The Biobrick prefix and suffix were applied to the gene

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) 1 pmid=16061392
 * 2) 2 pmid=16997527
 * 3) 3 pmid=10974125

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) 1 pmid=11738083
 * 2) 2 pmid=11911775
 * 3) 3 pmid=16997527
 * 4) 4 pmid=10974125

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) 1 pmid=16061392
 * 2) 2 pmid=16997527
 * 3) 3 pmid=10974125

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) 1 pmid=11738083
 * 2) 2 pmid=11911775
 * 3) 3 pmid=16997527
 * 4) 4 pmid=10974125

Codes and associated parts
{| border="2" cellpadding="2" style="text-align:left; background:black" ! Code ! Part ! Code ! Part ! Code ! Part ! Code ! Part ! Code ! Part
 * - style=background:springgreen
 * - style=background:orange
 * K143000 || || K143001 || 5' AmyE || K143002 || 3' AmyE || K143003 || 5' PyrD || K143004 || 3' PyrD
 * - style=background:orange
 * K143005 || 5' EpsE || K143006 || 3'EpsE || K143007 || || K143008 || || K143009 ||
 * - style=background:orange
 * K143010 || || K143011 ||  Promoter P43 || K143012 || Promoter Pveg || K143013 || Promoter Phyper-spank || K143014 || Promoter Pxyl
 * - style=background:orange
 * K143015 || Promoter Pctc || K143016 || Promoter PgsiB || K143017 || || K143018 || || K143019 ||
 * - style=background:orange
 * K143020 || || K143021 || gsiB RBS|| K143022 || spoVG RBS || K143023 || || K143024 ||
 * - style=background:orange
 * K143025 || || K143026 || || K143027 ||  || K143028 || || K143029 ||
 * - style=background:orange
 * K143030 || || K143031 || Aad 9 || K143032 || EpsE || K143033 || LacI || K143034 || LipA-EAK16
 * - style=background:orange
 * K143035 || LipA-Elastin || K143036 || XylR || K143037 || YtvA || K143038 || SacB-EAK16 || K143039 || SacB-Elastin
 * - style=background:orange
 * K143040 || || K143041 ||  || K143042 ||  || K143043 ||  || K143044 ||
 * - style=background:orange
 * K143045 || || K143046 ||  || K143047 ||  || K143048 || || K143049 ||
 * - style=background:powderblue
 * K143050 || P43-gsiB || K143051 || P43-spoVG || K143052 || Pveg-gsiB  || K143053 || Pveg-spoVG || K143054 || Phyperspank-gsiB
 * - style=background:powderblue
 * K143055 || Phyper-spank-spoVG || K143056 || Pxyl-gsiB || K143057 || Pxyl-spoVG || K143058 || Pctc-gsiB || K143059 || Pctc-spoVG
 * - style=background:powderblue
 * K143060 || PgsiB-gsiB || K143061 || PgsiB-spoVG || K143062 || CAT - Terminator || K143063 || Aad9 - Terminator || K143064 || LacI - Terminator
 * - style=background:powderblue
 * K143065 || XylR - Terminator || K143066 || Int Open || K143067 || RI Brick || K143068 || RI-Rep-Pi Brick || K143069 || RI-Ytva-Pl Brick
 * - style=background:powderblue
 * K143070 || Bs-PTC || K143071 || Int Close || K143072 || || K143073 || || K143074 ||
 * - style=background:powderblue
 * K143075 || || K143076 || || K143077 ||  || K143078 || || K143079 ||
 * - style=background:powderblue
 * K143080 || || K143081 || || K143082 ||  || K143083 || || K143084 ||
 * - style=background:powderblue
 * K143085 || || K143086 || || K143087 ||  || K143088 || || K143089 ||
 * - style=background:gold
 * K143090 || || K143091 || || K143092 ||  || K143093 || || K143094 ||
 * - style=background:gold
 * K143095 || || K143096 || || K143097 ||  || K143098 || || K143099 ||
 * -}

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