Team:WLC-Milwaukee/Parts

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Submitted Parts

<groupparts>iGEM013 WLC-Milwaukee</groupparts> BBa_K1175005 and K1175400 have not been sent to the registry
endo-1,4-beta-xylanase xynA from Bacillus Subtilis Subtilis 168

The endo-1,4-beta-xylanase gene xynA cleaves xylan polysaccharide chains to form shorter xylan chains. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168.

Usage and Biology

Xylan is a molecule similar to cellulose, and after cellulose the most abundant biomass material on earth. It is a major structural component of plant cell walls. Furthermore, xylan crosslinks with cellulose and other cell wall components, inhibiting access of cellulases (1). Xylose is the sugar monomer of xylan as glucose is to cellulose. Xylose cannot be used in the human body as a source of energy. Endo-1,4-beta-xylanase (xynA) breaks the xylan chains into shorter chains, and may be stearically hindered by side chains (2). A beta-xylanase such as Endo-1,4-beta xylanase may be used to degrade xylan to facilitate cellulase activity. Another use may be in conjunction with an exo-xylanase to efficiently break down xylan into xylose monomers (a pentose sugar).

Enzymatic Activity of Gene Product

As stated above, the function of the gene product is xylan degredation. The enzyme's catabolic activity results from endohydrolysis of 1,4-beta-D-xylosidic linkages in xylan molecules (4).

(1) http://newscenter.lbl.gov/feature-stories/2012/11/12/a-better-route-to-xylan/ (2) http://www.nutrex.be/sites/default/files/wysiwyg-upload/nutrase-xyla-nsp-enzyme.pdf (3) http://subtiwiki.uni-goettingen.de/wiki/index.php/XynA (4) http://www.uniprot.org/uniprot/P18429

endo-beta-1,3-1,4 glucanase (BglS) from Bacillus Subtilis 168

BglS This gene encodes an endo-beta-1,3-1,4-glucanase (bglS), which is from the bacterium Bacillus subtilis subtilis 168. The enzyme will hydrolyze and thereby cleave internal 1,4 linkages adjacent to 1,3 linkages.

BglS

Usage and Biology The substrates vulnerable to the bglS encoded enzyme are mixed linked beta-glucans. These glucans would have 1,3 and 1,4 beta linkages within the polysaccharide linking together the glucose monomers. Examples of these glucans can be found in oats, maize, and barley.

(1) http://mic.sgmjournals.org/content/141/2/281.long (2) http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1984.tb04259.x/pdf


beta-galacturonidase (YesZ) from Bacillus Subtilis 168

yesZ The enzyme acts on the terminal end of side chains of Rhamnogalacturonan I Pectin, releasing the free galactose. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168. The enzyme yesZ has beta-galactosidase (beta-galacturonidase) activity, cleaving (1→4)-β-D-galactans to produce single galactose molecules from RG I Pectin.

Usage and Biology Pectin is a component of the primary cell wall of plants. As galactose can be metabolized by the human body and by animals, it is an alternative to the usual target of nutrition/energy boosts in biotechnology, glucose.

(1) http://www.ncbi.nlm.nih.gov/pubmed/17449691 (2) http://subtiwiki.uni-goettingen.de/wiki/index.php/YesZ (3) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694227/ (4) http://aem.asm.org/content/73/12/3803 (5) http://www.tandfonline.com/doi/full/10.1080/15583724.2011.615962#tabModule


Strong pBAD promoter BBa_K206000 attached to SecI pump BBa_K215104

These are the three essential parts prtD, prtE, prtF, that make up the Type I secretion system from Erwinia chrysanthemi.This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The Pump is expressed from a strong constitutive promoter, BBa_k206000, and has the translational terminator BBa_B0014. In pSB1C3.

Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K215002]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system.

For full characterization data on this part, please see the University of Washington 2009 iGEM project page and Wisconsin Lutheran College 2013 iGEM project page.


BBa_K1175028 pBAD Promoter and SecI pump attached to Composite norvirus antibody BBa_K875004

These are the three essential parts prtD, prtE, prtF, that make up the Type I secretion system from Erwinia chrysanthemi.This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The Pump is expressed from a strong constitutive promoter, BBa_k206000, and has the translational terminator BBa_B0014. In pSB1C3.

Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K215002]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system.

This construct is designed for the expression of an already descibed engeneered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA-scFv 54.6 antinorovirus, Histidine tag (6HIS), Terminator (B0015).

The part combines a strongly expressed secretion pump with a antinoroviral antibody that is expressed in the outer-membrane can/will be combined with BBa_K1175012 A composite part formed between BBa_K206000 strong pBAD promoter, BBa_B0034 strong RBS, and BBa_K215001 a purification and secretion tag.

For full characterization data on this part, please see the University of Washington 2009 iGEM project page, Team Trieste iGEM 2012 and, Wisconsin Lutheran College 2013 iGEM project page.


BBa_K1175400

This combinatory part is a self contained secretion system that combines a secretion and purification tag, a tripartite pump that is specific to the sec tag, a antinorovirus particle like antibody, Tse2 toxin mediated horizontal gene transfer mechanism.

Tripartite SecI pump

These are the three essential parts prtD, prtE, prtF, that make up the Type I secretion system from Erwinia chrysanthemi.This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The Pump is expressed from a strong constitutive promoter, BBa_k206000, and has the translational terminator BBa_B0014. In pSB1C3. Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K1175012]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system.

Secretion and purification tag

This part BBa_K1175012 A composite part formed between BBa_K206000 strong pBAD promoter, BBa_B0034 strong RBS, and BBa_K215001 a purification and secretion tag; a secretion system designed by Washington iGEM team 2009 BBa_K215001. This tag can be used to secrete proteins of interest by inserting them into a specific NheI cut site flanked by C terminus His tag and an N terminus Secretion tag. Both tags can be removed by a TEV protease. The secretion tag is also specific to the tripartite pump.

Antinorovirus like particle antibody

This construct BBa_K875004 is designed for the expression of an already described engineered antinorovirus (NoV) monoclonal antibody (mAb 54.6) in fusion with LPP-OmpA. The antibody is expressed in a single chain fragment variable (scFv) format containing light (VL) and heavy (VH) variable domains separeted by a flexible peptide linker. It has already been reported that the scFv 54.6 binds a native recombinant NoV particles (VLPs) and inhibits VLP interaction with cells. LPP-OmpA functions as a leader sequence and an anchor to display the scFv ot the bacterial surface.The construct consistes of T5 Lac Operator (Bba_K875002), ribosomal binding site, LPP-OmpA-scFv 54.6 antinorovirus, Histidine tag (6HIS), Terminator (B0015).

T5cumate repressed Tse2 mediated, antihorizontal transfer mechanism

This construct BBa_K1175003 is a modification of the part designed by iGEM2012 Team Trieste it has a T5cumate inducible promoter suppressed by the CymR gene integrated genome into the chassis E.coli Nissle 1917. This part will not be suppressed by CymR in the bacterial cells that have taken up the plasmid.

WHAT: We are repurposing cellulolytic genes to increase bioavailable energy by breaking down cellulose into glucose, which can be used for humanitarian efforts, economic mobility and Industrial application

WHY: Increase bioavailable fuel sources for human consumption, economic growth and increased reliance on local resources.

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