IGEM:Imperial/2010/Parts/Registry Upload

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Contents

Registry Upload

All parts require a basic level of information regarding their sequence, use, design and function. To make information upload as efficient as possible it should all be collated here before submission.

Kirill will provide part numbers!

Necessary information

Copy the following tempalte to ensure that all the required information about each part has been gathered so it can be checked on Monday prior to uploading. An example of how this information should be filled in is included below.

Name: Part name goes here

Code: BBa_K???????

Sequence: ACGT etc.

Short: A short name/decsription of the part (Max. 60 characters inc. spaces)

Long: A thorough description of the part, including what function it has, where it is natively (ie. orignially - eg. E. coli genome locus XYZ) found and what context you have used it in if applicable. References where appropriate!

Source: Where did the part come from (eg. genome, vector). use references where possible!

Design: How was it made? Eg. PCR, Biobrick cloning from X and Y (Use <bbpart></bbpart> function if possible - it doesn't work here but will on the registry). If made by PCr, what enzyme and what primers - e-mail Chris if you don't have their sequences!

References: Very helpful if you have them!

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Example

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[2] and is still commonly used in vectors such as pDR111[3], pDL[4] 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[5]. 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[3, 6, 7]

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. . pmid:3019840. PubMed HubMed [1]
  2. Nakano S, Küster-Schöck E, Grossman AD, and Zuber P. . pmid:14597697. PubMed HubMed [2]
  3. Bacillus Genetic Stock Center [www.bgsc.org]

    [3]

  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.

    [4]

  5. Erwin KN, Nakano S, and Zuber P. . pmid:15937167. PubMed HubMed [5]
  6. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. . pmid:12169614. PubMed HubMed [6]
All Medline abstracts: PubMed HubMed

Parts

**K316012 - 3' K316000-sRBS-TEV**

Name: 3' coding, Enhanced LacI inducible promoter, synthetic RBS, TEV

Code: BBa_K316000

Sequence:

Short: 3' coding TEV protease under control of enhanced Lac operon

Long:

TEV protease S219P autocatalysis resistant variant

Introduction :

This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S [1]] between QG or QSDue to it’s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins.

Uses:

TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity [[2]] and potential to be used in vivo or in vitro applications.


Auto-inactivation

Wild type TEV protease also cleaves itself at Met 218 and Ser 219 [[3]] . This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease [[4]]

More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline)

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

Table I. Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. Enzyme Km (mM) kcat (s−1) kcat/Km (mM−1 s−1) Wild-type 0.061 ± 0.010 0.16 ± 0.01 2.62 ± 0.46 S219V* 0.041 ± 0.010 0.19 ± 0.01 4.63 ± 1.16 S219P* 0.066 ± 0.008 0.09 ± 0.01 1.36 ± 0.22

S219P* - virtually imperivious to autocatalysis S219V* - retains same activity as wild type

Full article can be seen here [[5]]



Source: TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg – eurofins www.eurofinsdna.com proprietary software.

Design: The part was produced by nucleotide synthesis by mwg – eurofins

References: Very helpful if you have them!

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**K316001 - Pveg**

Name: Pveg promoter

Code: BBa_K316001

Sequence: ACGT etc.

Short: pVeg Constitutive promoter for Veg locus from B. subtilis

Long: This part is identical to the sequence submitted by Imperial 2008 team, this part was produced from BBa_K143053 by PCR. PVeg is a constitutive promoter controlled by Sigma factor A. This promoter has two binding sites which leads to high expression of downstream genes. There is some evidence that the sporulation master regulator the spoOA can interact with pVeg although the mechanism is not known.

Source: PCR from existing biobrick K143053 using Pfu polymerase II

Design: PCR using Pfu polymerase to avoid mutations

References: Very helpful if you have them!

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**K316002 - dif**

Name: B. subtilis dif excision site

Code: BBa_K316002

Sequence:

Short: B. subtilis dif: sequence-specific recombinase target site

Long:

B. subtilis dif: sequence-specific recombinase recognition site

Cells with circular chromosomes, recombinatorial repair and homologous recombination can generate multimeric chromosomes [2]. ‘’Dif’’ sites are part of a system to ensure that multimeric chromosomes can be separated to monomers, which is required for proper sharing of genetic material between daughter cells. In ‘’B. subtilis’’ the tyrosine family recombinases such as RipX and CodV mediate the separation at 28-bp sequence Bs’’dif’’ [2]. The site-specific recombinases are able to recognize two directly repeated ’’dif’’ sites and excise the fragment flanked by the two sites [3].


http://partsregistry.org/Image:DifExcision.PNG

Figure 1. Removal of a specific gene from a genome integrated construct.

The site-specific recombinases, endogenous to ‘’B. subtilis’’ strains are able to recognise Bs’’dif’’ sites and recombine out the strand of DNA directly flanked by the two sites. Recombination leaves a single dif site.

The construct was previously engineered to homologously recobine into the genome of ‘’B. subtilis’’. Integration sequences such as amyE <bbpart>BBa_J143001</bbpart>, <bbpart>BBa_J143002</bbpart> can be used to achieve this.

Source: The dif sites were made by annealing synthestised oligoes.

Design: The dif site was made by oligos designed to make overhangs for EcoRI and SpeI ( and ) or XbaI and PstI ( and ) to be used in standard Biobrick or 3A cloning.

References: Very helpful if you have them!

References

  1. Sciochetti SA, Piggot PJ, and Blakely GW. . pmid:11208805. PubMed HubMed [1]
  2. Bloor AE and Cranenburgh RM. . pmid:16597952. PubMed HubMed [2]
All Medline abstracts: PubMed HubMedReferences

**K316003 - XylE-B0014**

Name: XylE, Double Terminator

Code: BBa_S04510

Sequence:

Short: XylE - catechol 2,3-dioxygenase from P.putida with terminator

Long: Catechol or catechol 2,3-dioxygenase + O(2) is converted by a ring cleavage into 2-hydroxymuconate semialdehyde which is the toxic and bright yellow-coloured substrate. This is a key enzyme in many (soil) bacterial species used for the degradation of aromatic compounds. The catechol 2,3-dioxygenase (pdb id: 1MPY[6]) used here itself originating from Pseudomonas putida is a homotetramer of C230 monomers. The tetramerization interactions position a ferrous ion critical for enzymatic activity. It has been deduced that intersubunit interaction is essential to produce a functioning enzyme after performing N and C terminal modifications on the monomer. Coming together the subunits generate an active site. The reaction itself takes place within seconds after the addition by Pasteur pipette or spraying of catechol at a 100mM stock solution diluted with DDH20 (used by our lab.) The toxic byproduct is thought to interfere with cell wall integrity and cellular machinery such that exposed cells gradually die.

Catechol 2,3-dioxygenase

Source: XylE was obtained from the registry <bbpart>BBa_K118021</bbpart>, the terminator is <bbpart>BBa_B0014</bbpart>. XylE is naturally found in Pseudomonas putida.

Design: The parts were put together using standard assembly [7]


== References: PubMed id: 10368270] http://www.ebi.ac.uk/pdbsum/1mpy Biochem. J. (2003) 371, 557–564 (Printed in Great Britain) 557 Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases Akiko OKUTA1, Kouhei OHNISHI2,3, Sakiko YAGAME and Shigeaki HARAYAMA Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan


</biblio> ==

**K316004 - J23101-XylE-B0014**

Name: Constitutive E-coli promoter, XylE, double terminator

Code: BBa_K316004

Sequence:

Short: Functional XylE under J23101 promoter, with B0014 terminator

Long: <bbpart>BBa_J23101</bbpart> combined with <bbpart>BBa_S04510</bbpart>for easy characterisation of gene activity (catechol breakdown into 2,4 hydroxyaminobutane yellow product) under a standard E.coli promoter

Source: Biobrick parts <bbpart>BBa_J23101</bbpart> and <bbpart>BBa_S04510</bbpart>

Design: Parts were assembled in two steps using standard biobrick assembly.

References: Very helpful if you have them!

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**K316005 - K316001-XylE-B0014**

Name: Pveg-XylE-Double Terminator

Code: BBa_K316005

Sequence:

Short: Functional XylE under Pveg promoter, with B0014 terminator

Long: Enables characterization and comparison with alternative promoters e.g. <bbpart>BBa_J23101</bbpart> Designed for characterisation of XylE in Bacillus subtilis where Pveg is becoming a standard promoter

Source: Biobrick parts <bbpart>BBa_J33204</bbpart> <bbpart>BBa_B0014</bbpart>

Design: Parts were assembled in two steps using standard biobrick assembly.

References: Very helpful if you have them!

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**K316006 - 5'hisGFP-XylE**

Name: 5'his tagged-GFP-XylE fusion protein

Code: BBa_K316006

Sequence:

Short: 5'His-tagged GFP linked to XylE by TEV cleavable linker

Long: Constructed to be combined with promoter and terminator. The GFP <bbpart>BBa_E0040</bbpart> is linked to XylE <bbpart>BBa_J33204</bbpart> monomer subunit by a GGGSGGGS linker with the aim to render the enzyme inactive, via preventing tetramerization (it’s functional form).

Please see ‘Part Design’ section for design considerations and parts used.

Source: Existing biobrick parts with modifications using PCR primer extension

Design: 'PCR primer extension - exact methods our wiki [8]

References: Very helpful if you have them!

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**K316007 - K143053-GFP-XylE**

Name: B. subtilis promoter Pveg and strong RBS spoVG <bbpart>BBa_K143053</bbpart>, 5'His tagged GFP-linker-XylE fusion protein

Code: BBa_K316007

Sequence:

Short: Cleavable GFP-XylE protein under Pveg promoter

Long: This construct is designed so that the XylE activity is substantially reduced untill such a time when a TEV protease is added to the system and transcribed. TEV protease cleavable linker is positioned between the two proteins. Once the linker is cleaved, XylE is free to tetramerise and assume full activity. GFP is His tagged at the 5' end to facilitate purificaiton for in-vitro assays.

Source: Pveg and spoVG biobricks <bbpart>BBa_K143053</bbpart> added to GFP-XylE construct <bbpart>BBa_K316004</bbpart>.

Design: Standard biobrick cloning of intermediary parts add part numbers <bbpart>BBa_K143053</bbpart>

References: Very helpful if you have them!

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**K316008 - K143053-5'hisGFP-linker-XylE-B0014**

Name: B. subtilis promoter Pveg and strong RBS spoVG <bbpart>BBa_K143053</bbpart>, 5'His tagged GFP-linker-XylE fusion protein <bbpart>BBa_K316005</bbpart>, double terminator <bbpart>BBa_B0014</bbpart>

Code: BBa_K316008

Sequence:

Short: Cleavable GFP-XylE fusion with Pveg promoter and terminator

Long: This construct is designed so that the XylE activity is substantially reduced untill such a time when a TEV protease is added to the system and transcribed. TEV protease cleavable linker is positioned between the two proteins. Once the linker is cleaved, XylE is free to tetramerise and assume full activity. GFP is His tagged at the 5' end to facilitate purificaiton for in-vitro assays. Terminator <bbpart>BBa_B0014</bbpart> has been added to comply with Biobrick standards. This particular terminator is stronger and is different from <bbpart>BBa_B0015</bbpart>.

Source: Existing Biobricks <bbpart>BBa_K143053</bbpart>, <bbpart>BBa_K316005</bbpart>, <bbpart>BBa_B0015</bbpart>

Design: Standard Biobrick assembly

References: Very helpful if you have them!

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**K316009 -TEV-sRBS-K316000 -K143053-5'hisGFP-linker-XylE-B0014**

Name: minus strand encoded - LacI inducible TEV and plus strand 5'his tagged-GFP-linker-XylE construct under constitutive Pveg promoter and spoVG RBS <bbpart>BBa_K143053</bbpart>

Code: BBa_K316009

Sequence:

Short: LacI inducible Fast Response Module, using cleavable XylE

Long:

5' his tagged GFP-TEV linker-XylE construct is pre-made in the cell under constitutive promoter. Both Pveg promoter and spoVG RBS are best suited for maximal expression in B.subtilis. While GFP is attached to the XylE monomer via the TEV cleavable linker, the catalytic activity is low. Transcription of TEV protease allows cleavage of the linker between GFP and XylE, thus XylE is free to tetramerise into a fully functional enzyme. XylE is then able to act as described in <bbpart>BBa_k316004</bbpart>

Please see 'Part Design' section for design considerations and parts used.

Source: Made from parts - <bbpart>BBa_k316012</bbpart> and <bbpart>BBa_k316008</bbpart>

Design: Standard biobrick assembly

References: Very helpful if you have them!

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K316010 -** 3' encoded K316000 - XylE**

Name: LacI inducible XylE

Code: BBa_K316010

Sequence:

Short: 3' strand coding XylE under LacI activation

Long: This part contains XylE under synthetic hyperspank promoter <bbpart>BBa_K143000</bbpart> LacI and IPTG are required for repression and activation of expression. As transformation of B subtilis often requires genomic integration, read-through from upstream genomic regions can become an issue. For the purposes of characterisation, the part was made on the minus strand to reduce background transcription. Read-through may provide an additional benefit as low quantities of anti-sence RNA may reduce basal expression sometimes seen in LacI systems. (although there is some evidence background is usually due to readthrough if have ref)

Source:

Design: the part contains a synthetic RBS optimised for high expression in B subtilis, calculated using RBS calculator [ link ]

References: Very helpful if you have them!

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K316011 - Flipped Xyle

Name: Flipped XylE

Code: BBa_K316011

Sequence:


Short: Reverse strand coding XylE

Long: This part contains XylE coding on the 3' strand. In many biological settings there may be read-through caused by upstream elements, however this can often be unidirectional. In settings where readthrough from 5' direction is expected to be much stronger than 3' direction, it may is advantageous to use 3' coding sequence instead of traditional 5'.

For the purposes of characterisation, the part was made on the minus strand to reduce background transcription. Read-through from 5' direction may provide an additional benefit. Low quantities of anti-sence RNA may be produced as the result of 5' readthrough, which may in turn reduce basal expression sometimes seen in LacI systems.

Source: This part is a modified existing biobrick <bbpart>BBa_J33204</bbpart>

Design: Part was designed using several rounds of PCR, using Pfu polymerase to avoid mutations. For more information, please check our wiki[9]

**K316000 - Pehs**

Name: Enhanced hyperspank promoter

Code: BBa_K316000

Sequence:

Short: 3' coding Enhanced LacI-hyperspank promoter

Long:

This part is a modified version of hyper-spank promoter for B.subtilis <bbpart>BBa_K143015</bbpart>. Hyper-spank promoter is repressed by transcriptional repressor LacI <bbpart>BBa_K143033</bbpart> and can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Constitutive expression of LacI is required for repression.

Promoter Design

The position and sequence of LacI binding was designed using existing knowledge. The stochastic nature of transcriptional repressors usually leads to background transcription. In order to minimise background the binding sites and the distance between them have been optimised.

Stronger binding

The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences. Perfectly symmetric binding sequence was shown to have10-fold higher binding compared to wild type sequences. The aattgtgagc gctcacaatt sequence has been shown to be optimal for LacI binding Muller 1996 Oehler 1994.

Optimal distance

Due to the tetrameric nature of LacI it can simulataneously bind to multiple regions in the genome. Binding at multple sites can produce much stronger repression (muller 1996) by increasing local LacI concentrations. Due to the helical nature of DNA the distance between the operator sites plays an important role in the strength of repression. Maximal repression at 70.5bp, second strongest at 92.5bp and third at 115.5bp


Source:

Design: Designed for minimal basal transcription by altering binding site sequence and distance.

References: Very helpful if you have them!

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**K316013 - PmeI cutting site**

Name: PmeI recognition sequence

Code: BBa_K316014

Sequence:

Short: 8bp recognition sequence for PmeI restriction endonuclease

Long:

Information about PmeI restriction endonuclease is available at [10]. The recognition site is a 8bp sequence GTTTAAAC. Pme produces a blunt cut after GTTT.

Source: This is a planning part. The sequence was combined with <bbpart>BBa_K143000</bbpart> to produce <bbpart>BBa_K143014</bbpart> in construction of B. subtilis genome integration vectors

Design: This is a planning part. The sequence was produced from single stranded primer oligos.

References: Very helpful if you have them!

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**K316014 - K316000-K316013**

Name: Dif Pme

Code: BBa_K316014

Sequence:

Short: Dif sequence followed by PmeI recognition site

Long:

This composite part of <bbpart>BBa_K143000</bbpart> and <bbpart>BBa_K143013</bbpart>. The dif site can be used in conjunction with another dif site in another part of the vector to remove a sequence between the two dif sites. PmeI site can be used for blunt end cloning of a DNA sequence behind the dif site.

Source: oligonucleotide synthesis

Design: This part was designed to be cloned using standard biobrick methods. Two single stranded, synthetic oligos were annealed to produce double stranded DNA sequence with single stranded overhangs identical to the product of digestion by XbaI and SpeI. Thus compatible with biobrick cloning methods.

References: Very helpful if you have them!

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**K316015 - ComD**

Name: ComD Receptor

Code: BBa_K316015

Sequence:

Short: ComD receptor for CSP-1 (S. pneumoniae). Phosphorylates ComE

Long: ComD is the histidine kinase of a two-component signal transduction signaling pathway in a Streptococcus pneumoniae quorum sensing system. It detects the linear autoinducing peptide (AIP) called Competence-Stimulating Peptide-1 (CSP-1) which is coded for by the ComC gene. Upon detection of CSP-1, ComD will autophosphoylate and then phosphorylate and activate the response regulator, ComE

Source: Streptococcus pneumoniae, AAC44896.1, codon optimised for expression in B.subtilis

Design: the part contains a synthetic RBS optimised for high expression in B subtilis, calculated using RBS calculator [ link ]

References:

Info on threshold levels of CSP-1 etc [[11]]

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**K316016 -ComE**

Name: LacI inducible XylE

Code: BBa_K316016

Sequence:

Short: Activates transcription of target genes when phosphorylated.

Long: ComE is the response regulator of a two-component signal transduction signaling pathway in a Streptococcus pneumoniae quorum sensing system. It is phosphorylate and activated by ligand-bound ComD. It will then induce transcription of any gene which has a ComE binding site in its promoter.


Source: Streptococcus pneumoniae, AAC44897, codon optimised for expression in B. subtilis

Design: Synthetic RBS to optimise translation in B. subtilis as part of poly-mRNA containing ComD upstream

References:

Designing a ComE binding site: [[12]] Dimerisation of ComE and more info on binding site: [[13]]


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**K316017 -TEV+B316000**

Name: LacI inducible TEV protease


Short

LacI operator controlled TEV protease

Long


TEV protease S219P autocatalysis resistant variant <bbpart>BBa_K316012</bbpart>. This part had been reversed for the 3' strand in order to reduce any read-through that may be caused by upstream elements. Expression of TEV protease is under control of enhanced hyperspank promoter <bbpart>BBa_K316000</bbpart>.

Please note all the sequences of this part are reversed to code on 3' strand


The part was produced by nucleotide synthesis by mwg – eurofins. The synthetic RBS was designed to work with <bbpart>BBa_K316000</bbpart>.


TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg – eurofins [www.eurofinsdna.com] proprietary software.

**K316018 -Com CDE forward

Name: ComE responsive promoter


Short

ComE responsive promoter

Long

ComE binds to a specific sequence leading to activation of trancription.


The part was produced by nucleotide synthesis by mwg – eurofins. The synthetic RBS was designed to work with <bbpart>BBa_K316000</bbpart>.


TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg – eurofins [www.eurofinsdna.com] proprietary software.


**K316019 -Com CDE reverse

Name: ComE responsive promoter


Short

ComE responsive promoter

Long

ComE binds to a specific sequence leading to activation of trancription.


The part was produced by nucleotide synthesis by mwg – eurofins. The synthetic RBS was designed to work with <bbpart>BBa_K316000</bbpart>.


TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg – eurofins [www.eurofinsdna.com] proprietary software.

**K316020 -pyrD complete vector-K143008-K316002-K143053-K143065-K316014-K143009**

Name: B. subtilis genome integration vector into pyrD locus

Code: BBa_K316020

Sequence:

Short: B. subtilis genome integration vector into pyrD locus

Long:

PyrD This vector has been designed using the Pyrd 5' <bbpart>BBa_K143001</bbpart> and 3' <bbpart>BBa_K143002</bbpart> integration sequences for integration into B. subtilis genome. Insertion into the pyrD locus provides a negative selection marker as the bacterium will no longer be able to synthesise uracil. Thus medium supplement of 40ug/ml is required for growth. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments.

Spectinomycin Resistance This vector also contains a positive selection marker, flanked by two dif sites. Aad9 <bbpart>BBa_K143065</bbpart> provides resistance to spectinomycin antibiotic. Dif <bbpart>BBa_K316002</bbpart> sites allow excision of the antibiotic marker after integration, thus allowing the same marker to be used again or as a precaution against horizontal gene transfer.

Blunt end cloning site PmeI restriction site <bbpart>BBa_K316013</bbpart> is designed as a cloning site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once.


Please see ‘Part Design’ section for design considerations and parts used.

Source: Existing biobricks, <bbpart>BBa_K143008</bbpart>, <bbpart>BBa_K316002</bbpart>, <bbpart>BBa_K143053</bbpart> <bbpart>BBa_K143065</bbpart> <bbpart>BBa_K316014</bbpart> <bbpart>BBa_K143009</bbpart>

Design: Using standard assembly of biobricks

BBa_K143008 BBa_K316002 BBa_K143053 BBa_K143065 BBa_K316014 BBa_K143009

References: Very helpful if you have them!

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**BBa_K316021 BBa_K143053 BBa_K143065**

Name: Pveg-spoVG-Spec-B0015

Code: BBa_K316021

Sequence:

Short:

Spectinomycin resistance with promoter for B.subtilis

Long:

For reliable expression in B. subtilis the strongest constitutive promoter Pveg and strong ribosome binding site spoVG <bbpart>BBa_K143053</bbpart> were combined with spectinomycin resistance gene with terminator <bbpart>BBa_J143065</bbpart>.

Please see ‘Part Design’ section for design considerations and parts used.


Source:

Existing biobricks <bbpart>BBa_K143053</bbpart>, <bbpart>BBa_J143065</bbpart>

Design considerations:

Standard biobrick assembly [14]

References: Very helpful if you have them!

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**K316022 - K143001-k316002-K143053-K143065-K316002-K316014-K143002**

Name:amyE complete vector 5'amyE-diff-Pveg-spoVG-CAT-B0015-diff-PmeI-3'amyE

Code: BBa_K316022

Sequence:

Short:

B.subtilis transformation vector, targets Amylase locus

Long:

This vector has been designed using the amyE 5' and 3' integration sequences for integration into B.subtilis genome

AmyE locus This vector has been designed using the amyE 5' <bbpart>BBa_K143008</bbpart> and 3 <bbpart>BBa_K143009</bbpart>' integration sequences for integration into B. subtilis genome. Insertion into the amyE locus provides a selection marker as the bacterium will no longer be able to breakdown starch. An iodine assay can be used to confirm integration. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments.

Chloramphenicol Resistance This vector also contains a positive selection marker, flanked by two dif sites. Chloramphenicol acetyltransferase <bbpart>BBa_J31005</bbpart> provides resistance to chloramphenicol antibiotic. Dif <bbpart>BBa_K316002</bbpart> sites allow excision of the antibiotic marker after integration, thus allowing the same marker to be used again or as a precaution against horizontal gene transfer.

Blunt end cloning site PmeI restriction site <bbpart>BBa_K316013</bbpart> is designed as a cloning site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once.


Please see ‘Part Design’ section for design considerations and parts used.



Source: Existing biobricks, <bbpart>BBa_K143070</bbpart>, <bbpart>BBa_K316002</bbpart>, <bbpart>BBa_K316014</bbpart> <bbpart>BBa_K143002</bbpart>

Design: the part contains a synthetic RBS optimised for high expression in B subtilis, calculated using RBS calculator [ link ]

BBa_K143001 BBa_K316002 BBa_K143052 BBa_J31005 BBa_K316014 BBa_K143002

BBa_K143001 BBa_K143052 BBa_J31005 BBa_K143002

References: Very helpful if you have them!

  1. []

**K316023 - K143070 - K143064**

Name: 5'amye-Pveg-spoVG-CAT-B0015

Code: BBa_K316023

Sequence:

Short:

Chloraphenicol resistance gene with 5' amyE sequence

Long:

Chloramphenicol acetyltransferase (CAT) <bbpart>BBa_K143031</bbpart> confers resistance to chloramphenicol, a common lab selection antibiotic. The 5' amyE <bbpart>BBa_K143001</bbpart> integration sequence allows integration into B. subtilis genome, which disrupts ability to breakdown starch. In order for integration to occur, 3' amyE integration sequence <bbpart>BBa_K143002</bbpart> is also required.

This part can be used as the 5' start of an amyE integration vector, the genes of interest can then be attached to the 3' end, followed by the 3' amye integratio sequwnce <bbpart>BBa_K143002</bbpart>.

Please see ‘Part Design’ section for design considerations and parts used.

Source: Biobrick parts <bbpart>BBa_K143070</bbpart> (which contains <bbpart>BBa_K143001</bbpart>, <bbpart>BBa_K143012</bbpart>, <bbpart>BBa_K143021</bbpart>) and <bbpart>BBa_K143064</bbpart> (which contains <bbpart>BBa_K143031</bbpart>, <bbpart>BBa_B0015</bbpart>)

Design: Parts combined by standard biobrick assembly [15]

References: Very helpful if you have them!

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**K316024 - K316023+dif - K143070-K143064**

Name: 5'amye-diff-Pveg-spoVG-CAT-B0015

Code: BBa_K316024

Sequence:

Short:

Chloraphenicol resistance gene with dif and 5' amyE sequence

Long:

This part is identical to <bbpart>BBa_K143023</bbpart> except for the dif site<bbpart>BBa_K3160</bbpart> integrated behind the 5' amye <bbpart>BBa_K143001</bbpart> integration sequence.

Chloramphenicol acetyltransferase (CAT) <bbpart>BBa_J31005</bbpart> confers resistance to chloramphenicol, a common lab selection antibiotic. The 5' amyE <bbpart>BBa_K143001</bbpart> integration sequence allows integration into B. subtilis genome, which disrupts ability to breakdown starch. In order for integration to occur, 3' amyE integration sequence <bbpart>BBa_K143002</bbpart> is also required.

This part can be used as the 5' start of an amyE integration vector, the genes of interest can then be attached to the 3' end, followed by the 3' amye integratio sequwnce <bbpart>BBa_K143002</bbpart>.

Please see ‘Part Design’ section for design considerations and parts used.

Source: Biobrick parts <bbpart>BBa_K143070</bbpart> (which contains <bbpart>BBa_K143001</bbpart>, <bbpart>BBa_K143012</bbpart>, <bbpart>BBa_K143021</bbpart>) and <bbpart>BBa_K143064</bbpart> (which contains <bbpart>BBa_K143031</bbpart>, <bbpart>BBa_B0015</bbpart>)

Design: 'Reverse PCR' was used to amplify the whole vector, while adding the dif sites by primer extension. Pfu polymerase was used to ensure error-free replication.

References: Very helpful if you have them!

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K316025 - diff-K143002

Name: oh noes!?!!?!!!! not this one!!!

Dif-PmeI -3' amyE integration sequence

Code: BBa_K316025

Sequence:

Short:

Dif-PmeI site with 3' amyE integration sequence

Long:

This is the 3' part of B. subtilis genome integration vectors <bbpart>BBa_K316022</bbpart> and <bbpart>BBa_K316027</bbpart>. The 3' amyE integration sequence <bbpart>BBa_K143002</bbpart> for integration into B.subtilis genome was combined with a dif and PmeI recognition site <bbpart>BBa_K3160014</bbpart>.


Source: Biobrick <bbpart>BBa_K143002</bbpart> and synthetic oligos <bbpart>BBa_K316014</bbpart>

Design: Standard biobrick assembly

References: Very helpful if you have them!

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**K316027 -LacI testing vector K143001-k316002-K143053-K143065-K143053-K143062- K316002-K316014-K143002**

Name:amyE complete vector 5'amyE-diff-Pveg-spoVG-CAT-B0015-diff-PmeI-3'amyE

Code: BBa_K316022

Sequence:

Short:

B.subtilis transformation vector with LacI, targets Amylase locus

Long:

This vector has been designed using the amyE 5' and 3' integration sequences for integration into B.subtilis genome

AmyE locus This vector has been designed using the amyE 5' <bbpart>BBa_K143008</bbpart> and 3 <bbpart>BBa_K143009</bbpart>' integration sequences for integration into B. subtilis genome. Insertion into the amyE locus provides a selection marker as the bacterium will no longer be able to breakdown starch. An iodine assay can be used to confirm integration. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments.

Chloramphenicol Resistance This vector also contains a positive selection marker, flanked by two dif sites. Chloramphenicol acetyltransferase <bbpart>BBa_J31005</bbpart> provides resistance to chloramphenicol antibiotic. Dif <bbpart>BBa_K316002</bbpart> sites allow excision of the antibiotic marker after integration, thus allowing the same marker to be used again or as a precaution against horizontal gene transfer.

Blunt end cloning site PmeI restriction site <bbpart>BBa_K316013</bbpart> is designed as a cloning site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once.


For more information about our project please visit our wiki [16] or take the tour [17] to learn more about the project.


Source: Existing biobricks, <bbpart>BBa_K143070</bbpart>, <bbpart>BBa_K316002</bbpart>, <bbpart>BBa_K316014</bbpart> <bbpart>BBa_K143002</bbpart>

Design: This part is designed for integration into B. subtilis' genome at amyE locus, it contains Spectinomycin resistance selection marker and constitutively expresses LacI. PmeI can be used to ligate a gene of interest using blunt ended methods.

BBa_K143001 BBa_K316002 BBa_K143053 K143065 BBa_K143053 BBa_K1430562 BBa_K316014 BBa_K143002



References: Very helpful if you have them!

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**K316030 - LytC**

Name:

Code: BBa_K316030

Sequence:

Short: LytC cell wall anchoring protein for B. subtilis

Long: The cell wall binding domin of lytC, a N-acetylmuramoyl-L-alanine amidase of B. subtilis, was isolated to be used as a cell wall anchor. As demonstrated by Kobayashi et al. 2000 this domin can be used to target catalytic domians of other proteins as well as peptides to the cell wall of B. subtilis whilst leaving their functionality intact.

Source: Genome PCR from B. subtilis genome (strain)

Design: Pfu DNA polymerase was used to minimise PCR errors

References: Very helpful if you have them!

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**K316031 - K316001-LytC**

Name:

Code: BBa_K316031

Sequence:

Short: LytC cell wall anchoring protein with Pveg promoter

Long:

promoter - add links to both k316001 and K143012

The cell wall binding domin of lytC, a N-acetylmuramoyl-L-alanine amidase of B. subtilis, was isolated to be used as a cell wall anchor. As demonstrated by Kobayashi et al. 2000 this domin can be used to target catalytic domians of other proteins as well as peptides to the cell wall of B. subtilis whilst leaving their functionality intact.

Source: Genome PCR from B. subtilis genome (strain)

Design: Pfu DNA polymerase was used to minimise PCR errors

References: Very helpful if you have them!

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**K316031 - LytC with linker1**

Name: LytC-linker1-TEV cleavable site-ComC

Code: BBa_K316031

Sequence:

Short: B. subtilis Cell wall biniding domain with cleavable linker

Long: We designed a protein that carries our signal peptide ComC out of the cell where a protease has access to it. The protease can then proceed to cleave ComC off the protein, allowing quorum sensing via the the ComCDE system to take place. One big problem we have to overcome is the cell wall that will obstruct the protease’s access to the protein carrying the signal peptide. The Detection module consists of a cell wall anchor, a signal peptide called ComC, and a linker that connects the two and is specifically designed to be cleaved by the protease we want to detect

Source: Genome PCR from B. subtilis genome (strain) with linker-TEV cleavable site-ComC synthesised and attached via a PmeI site within LytC gene

Design: Pfu DNA polymerase was used to minimise PCR errors

References: Very helpful if you have them!

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K316032 - LytC

Name:

Code: BBa_K316032

Sequence:

Short: 3' strand encoded XylE under LacI activation

Long: The cell wall binding domin of lytC, a N-acetylmuramoyl-L-alanine amidase of B. subtilis, was isolated to be used as a cell wall anchor. As demonstrated by Kobayashi et al. 2000 this domin can be used to target catalytic domians of other proteins as well as peptides to the cell wall of B. subtilis whilst leaving their functionality intact.

Source: Genome PCR from B. subtilis genome (strain)

Design: Pfu DNA polymerase was used to minimise PCR errors

References: Very helpful if you have them!

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K316033 - LytC

Name: LytC – Glycin Linker – TEV Cleavage Site – His-Tag – Stop Codon

Code: BBa_K316033

Sequence:

Short: 3' strand encoded XylE under LacI activation

Long:

Introduction: This part was used to link the cell wall binding domain (CWB) of LytC, used in the detection module, with the quorum sensing peptide (AIP) as well as providing a cleavage site for a protease we want to detect. LytC: The part carries part of LytC on its 5’ end. This was used to ligate the linker with LytC via an internal ACCI restriction site that occurs naturally (Link to LytC sequence). Glycin Linker: The Linker separates the CWB and the AIP and creates space for the protease to access the cleavage site; it consists of two main sections. The first six amino acids (SRGSRA) were suggested to be used specifically with LytC (Yamamoto et al. 2003). The second section consists of a several glycin residues. TEV Cleavage Site: This sequence forms the 3’ end of the linker and is directly attached to the 5’ end of the AIP. It is 18 amino acids (GGGGENLYFQGGKLGGGG) long and was designed to be efficiently cleaved by the TEV protease, as well as being codon-optimised for expression in B. subtilis. His-Tag: To be able to purify the protein for testing, we attached a His-Tag on our linker-AIP peptide. As it would probably interfere with recognition of the AIP by the receptor it has to be removed from the final construct. Stop Codon: In order to end translation a double stop codon was put in place.


Source: Genome PCR from B. subtilis genome (strain)

Design: Pfu DNA polymerase was used to minimise PCR errors

References: Very helpful if you have them!

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K316034 - LytC

[2]

Pveg-spoVG-LytC-Helical Linker-Elastase Cleavage Site-AIP-His Tag BBa_K316037

Introduction: This part, our detection module, was designed to anchor a signal peptide (AIP) to the cell wall of B. subtilis which is released upon cleave of the part by a protease, in this case the cercarial elastase (UniProt, MEROPS), to activate our output. The part is fully assembled and ready to be expressed in a prokaryotic cell as it contains a promoter we characterised, an optimised RBS and a double stop codon. The protein product includes a cell wall binding domain (CWB) of LytC, BBa_K316030 a linker that has been designed to carry a protease cleave site and the AIP BBa_K316047. This part is essential for the great modularity of our system, as a change of just 4 amino acids can be used to confer specificity to another protease see our Software Tool. 


Pveg-spoVG: This was build in 2008 by the Imperial College iGEM team BBa_K143053.


LytC: The CWB is on the 5’ end and anchors the detection module to the cell wall of B. subtilis. The CWB was separately submitted as biobrick BBa_K316030.



Helical Linker: The Linker separates the CWB and the AIP and creates space for the protease to access the cleavage site; it consists of two main sections. The first six amino acids (SRGSRA) were suggested to be used specifically with LytC Yamamoto et al. 2003. The second section consists of a helical amino acid sequence that is stiff and prevents interaction of the AIP with its receptor and thus false positive activation of the output system. It was specifically designed to separate protein domains Arai et al. 2001.



Elastase Cleavage Site: This sequence forms the 3’ end of the linker and is directly attached to the 5’ end of the AIP. It is four amino acids (SWPL) long and was designed to be efficiently cleaved by the schistosoma cercarial elastase Salter et al. 2002.


AIP: The linear auto-inducing peptide (AIP) from the Streptococcus pneumonia ComCDE system, called Competence-Stimulating Peptide-1 (CSP-1 or ComC) . Upon cleavage it is free to diffuse and bind it's receptor ComD BBa_K316015. Signalling cascade from ComD is then able to activate transcription of genes under specific promoter sequences.


His-Tag: To be able to purify the protein for testing, we attached a His-Tag on our linker-AIP peptide. As it would probably interfere with recognition of the AIP by the receptor it has to be removed from the final construct. 


Stop Codon: In order to end translation a double stop codon was put in place.

K316040 - Experiment 2

K316040 - Experiment 6

XylE

Part Characterisation

Due to technical limitations, to measure kinetic parameters of XylE is to lyse cells and . In this experiment cell lysate was assayed with increasing catechol concentrations. The rate at which the yellow product appears is directly proportional to the velocity of the reaction. The rate reaction was monitored by measuring color output of the reaction in the plate reader.

Cell lysate was tested for dioxygenase activity to determine appropriate dilutions for the assay. The cell lysate was obtained from a 100ml overnight culture and diluted by a factor of 20 to obtain a suitable concentration of total enzyme for the plate reader assay. The concentrations of catechol used were 1, 2, 5, 10, 25, 50 mM.

Data collected was used to construct the Michaelis-Menten curve for the in vitro kinetics of XylE in cell lysate.


Figure I. Michaelis-Menten curve was drawn using velocity values calculated from the slope at the initial stages of the reaction, as this is the only time when substrate concentration values are accurate. The plot was delineated by non-linear regression analysis using GraFit software tool[18]. The calculated Km is 0.71mM catechol (with a Vmax of 3.37 in O.D. arbitrary units for this dilution of cell lysate).

For more detailed information, please check our wiki [19]



References

  1. Salis HM, Mirsky EA, and Voigt CA. . pmid:19801975. PubMed HubMed [1]
  2. http://voigtlab.ucsf.edu/software/

    [2]

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