Gluten digestion enzyme plasmid.

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This page is for Guy's research.

Stable/Genomic encoding

http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023

I am currently viewing Yeast integrative plasmid,(YIp), whhich are yeast vectors that utilize integration into the yeast chromosome for survival and are used when studying a DNA gene. http://en.wikipedia.org/wiki/Yeast_artificial_chromosome http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.37.100183.001345 http://www.pnas.org/content/75/4/1929.full.pdf http://nar.oxfordjournals.org/content/29/12/e59.full http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf (7.1.3) https://static.igem.org/mediawiki/2008/4/4e/JHU_0708_paper_Shuttle_Vectors_with_Multiple_Unique_Restriction_Sites.pdf

Vectors derived from the 2mm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2mm plasmid, others include just the 2mm origin of replication. An example of the latter type is YEp13 (Figure 7.3). YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector. As well as the 2mm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli. There are several lines of reasoning behind the use of shuttle vectors. One is that it may be difficult to recover the recombinant DNA molecule from a transformed yeast colony. This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes (p. 135), purification may be impossible.This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing. The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast (Figure 7.4). (1)

(1) Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene (Figure 7.6(a)). This gene codes for orotidine-5¢-phosphate decarboxylase (an enzyme that catalyses one of the steps in the biosynthesis pathway for pyrimidine nucleotides) and is used as a selectable marker in exactly the same way as LEU2. A YIp cannot replicate as a plasmid as it does not contain any parts of the 2mm plasmid, and instead depends for its survival on integration into yeast chromosomal DNA. Integration occurs just as described for a YEp (Figure 7.5). (2) Yeast replicative plasmids (YRps) are able to multiply as independent plasmids because they carry a chromosomal DNA sequence that includes an origin of replication. Replication origins are known to be located very close to several yeast genes, including one or two which can be used as selectable markers. YRp7 (Figure 7.6(b)) is an example of a replicative plasmid. It is made up of pBR322 plus the yeast gene TRP1. This gene, which is involved in tryptophan biosynthesis, is located adjacent to a chromosomal origin of replication.The yeast DNA fragment present in YRp7 contains both TRP1 and the origin. (1)

1. "Chapter 7 Cloning Vectors." Gene Cloning. N.p.: n.p., n.d. 135-37. Http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf. 22 Aug. 2005. Web.


iGEM Gluten Metabolism

iGEM Gluten Metabolism Project

When answering questions, please include citations

General Background

gliadin - wheat

hordein - barley

  1. What is gluten?

        -Gluten is the composite of a gliadin and a glutelin, which is conjoined with starch in the endosperm of various grass-related grains. The prolamin and glutelin from wheat (gliadin, which is alcohol-soluble, and glutenin, which is only soluble in dilute acids or alkalis) constitute about 80% of the protein contained in wheat seed.

http://en.wikipedia.org/wiki/Gluten

  1. What proteins cause celiac disease?

-While it is not entirely known what causes celiac disease, it has been theorized that certain peptide strands within gluten cause the immune system to recognize the gluten as harmful, therefore attacking the villi lining the intestines. (villi are responsible for absorbing nutrients). More specifically, there is a recurring sequence within the gluten that appears to be a root cause of the body’s reaction to gluten (PQPQLP). This sequence is found in multiple forms of gluten including gliadin, hordein, and each component’s sublevels. Studies have been conducted that posit that if this recurring sequence is systematically broken down, then the intestine may have a much greater ability to absorb incoming nutrients (i.e. food products containing gluten).        

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001280/

  1. How broken down do these proteins need to be to qualify as gluten-free?

-the oligopeptides that cause toxic reactions in Celiac patients need to be broken down in order to be digested with minimal reaction. Our proposed enzyme would cut the recurring sequence of PQPQLP into segments, therefore making it non-reactive to patients with Celiac disease. “oligopeptides are cleaved into fragments, thereby preventing the disease-causing toxicity”

For a more in depth analysis read this patent http://www.patentstorm.us/patents/7534426/description.html

  1. What protease do we want to express?

        Kumamolisin-As_N291D

  1. Why this protease?

-A mutated Kumamolisin-As enzyme aimed to break down gluten by increased activity with the PQLP peptide, an antigenic epitope in gliadin. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As_N291D so promising for the development of a pill (and beer!) for gluten intolerance.

https://2011.igem.org/Team:Washington/Celiacs/Background

  1. What glutens are found in beer/barley?

        -Hordein is the gluten component typically found in barley brewed beers. It has four main subcategories including B-hordein (70%), C-hordein (20%), and D and Gamma (remaining %).

This publication is great for info on the connection between barley brewed beer and Hordein

http://pubs.acs.org/doi/pdf/10.1021/pr2008434

In order for a beer to be Gluten free by industry standards, it must have <20 ppm gluten. But maybe the standard will change if Glute breakdown is considered?

http://www.glutenfreestandards.com/#!our-standard

General Laboratory Tools

  1. What cell lines do we have?  What are the advantages/disadvantages of each line?

        DH5 alpha

        Top10

        BL21

        Competent cell lines Steven

                Protocol for making competent cells?

        1) DH5alpha - bought, Invitrogen

Ethan - identify plasmids and amplify

miniprep

midi/maxiprep?

Steve/Ryan protease activity assay

        

Prokaryotic System Ethan

        what strain of E. coli? DH5a

        what promoter should we use to drive expression (all constitutive)?

  1. BBa-J23100 (strong)- http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100
  2. BBa-J23108 (medium) - http://partsregistry.org/Part:BBa_J23108
  3. BBa-J23109 (weak) - http://partsregistry.org/Part:BBa_J23109

        what terminator sequence?

                BBa-B0015  - http://partsregistry.org/Part:BBa_B0015

        what ribosome binding sites

                Constitutive - http://partsregistry.org/Part:BBa_B0034

        plasmid?

                http://partsregistry.org/Part:BBa_J13002

        can we get the protein to be secreted?

                Possibly - via a Type I C-terminal a-haemolysin secretion sequence on the C-terminus of the ORF

        can we track expression of the protein by a mechanism other than activity?

Characterization of protease

what do we need to get develop the functional assay for protease activity here

        what components do we need, where should we get them, and how much do they cost?

        how relevant is this assay to gluten metabolism

                evidence?

can we get an immunoassay for gluten levels in a solution?

        will the assay work well with hordein? gliadin asl?

variables of enzyme activity

        1) pH

                what range of pH is found in beer?

 - At the start of the brewing process, the pH of beer is 5.0-5.5, and usually ends around 4.0. Most beers are bottled at a pH of 4.0. The average pH drop during the brewing process is around 1.0.

                        what is the source of this information?

                                http://corrosion-doctors.org/Food-Industry/Beer-corrosion.htm

                                http://en.allexperts.com/q/Beer-2269/fermenting.htm

                                http://www.probrewer.com/vbulletin/archive/index.php/t-3957.html

        2) alcohol content -Dave

        3) temperature -Dave

                ale yeast?

-Ale fermentation typically occurs between 68 and 72 degrees fahrenheit

                lager yeast?

                -Lager fermentation typically occurs between 45 and 55 degrees fahrenheit

http://www.byo.com/stories/issue/article/issues/260-marchapril-2009/1923-controlling-fermentation-temperature-techniques

        4) how does osmolarity influence enzyme behavior -Dave

Yeast System

  1. What yeast are we using?

Saccharomyces cerevisiae Safale US-05

  1. homothallic or heterothallic?
  1. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating (heterothallic) may predominate in natural yeast populations, while mother-daughter mating (homothallic) might not be as significant as was considered

http://www.ncbi.nlm.nih.gov/pubmed/20002587

  1. diploid or haploid?
  1. Cells in the diploid phase are more resistant to harsh environmental conditions. When diploid cells begin to run out of food, they undergo meiosis, forming four haploid spores in an ascus (Saccharomyces cerevisiae belongs to the ascomycetes.)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/Y/Yeast.html

  1. Codon bias from prokaryotic system?  Dave

        The issue in Codon bias is that different organisms can produce different RNA's much easier. If the RNA used in a codon is uncommon, the plasmid is likely to die off. http://www.annualreviews.org/doi/pdf/10.1146/annurev.genet.42.110807.091442

        In particular, if a Codon is not common in a given organism, then the odds of replication decrease greatly.

  1. Are there enough issues that we need to recode our protease sequence? According to these two analyses, there are several codons used in Kumamolisin which are uncommon in yeast (occur <15% of the times the corresponding nucleic acid is encoded).

http://www.genscript.com/cgi-bin/tools/rare_codon_analysis

http://www.jcat.de/

(for both of these, copy in the Kumamolisin sequence from: http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 by clicking Get selected sequence on the bottom right).

An optimized sequence is given out by the jcat application, and also at: http://www.encorbio.com/protocols/Codon.htm.  The encorbio tools requests an amino acid input, which can be given by input the Kumamolisin sequence into: http://www.vivo.colostate.edu/molkit/translate/index.html .

The JCat and encorbio tools produce optimized sequences which are similar, but do not align exactly.  Both appear to be well-written algroithms, however significently more information is available on the JCat program, for this reason I recommend using the sequence this protein produced: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1160137/?tool=pubmed 

Information on the CAI rating: http://bioinformatics.oxfordjournals.org/content/19/16/2005.long 

  1. How would we accomplish this?  

Commercial synthesis does not appear to be an option, as chains are usually at most 200 bp (http://ac.els-cdn.com/S0040402001887524/1-s2.0-S0040402001887524-main.pdf?_tid=5674454e4d47830f0d55bf00ce2d88f9&acdnat=1339189034_de71a843d55cc459316c95f664294854 ), yet Kumamolisin is 1701bp.  However, if this is a route we want to explore, it’s worth contacting http://www.pmf.colostate.edu/genomics_pricing.html and verifying that this is the case.  Commercial synthesis would also be very expensive if it is possible, the price would be ~$400.  

Codon bias is best addressed by choosing the appropriate plasmid to fuse Kumamolisin to.  This can help maximize the odds that the plasmids are replicated in the yeast.  The combination of the plasmid and Kumamolisin can be analyzed using the above tools before the attempt in yeast.

  1. vector for regulated expression?
  2. vector for constitutive expression?
  3. vector for genomic insertion/integration? -Guy/Ethan
  1. Custom HR site - http://partsregistry.org/Part:BBa_K300001
  2. TRP1 -  http://partsregistry.org/Part:BBa_K165061
  3. LEU2 (works) - http://partsregistry.org/Part:BBa_K165062
  4. URA3 - http://partsregistry.org/Part:BBa_K165063
  5. HIS3 - http://partsregistry.org/Part:BBa_K165060
  6. ADE4 (works) - http://partsregistry.org/Part:BBa_K319043
  7. TRP1 (Lucas Argueso) - http://addgene.org/vector-database/3969/
  8. pCM189 (Lucas Argueso) centrimeric- http://web.uni-frankfurt.de/fb15/mikro/euroscarf/data/P30326.html
  9. pCM190 (Lucas Argueso) episomal- http://web.uni-frankfurt.de/fb15/mikro/euroscarf/data/P30327.html

Vectors derived from the 2mm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2mm plasmid, others include just the 2mm origin of replication. An example of the latter type is YEp13. YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector. As well as the 2mm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli. There are several lines of reasoning behind the use of shuttle vectors. One is that it may be difficult to recover the recombinant DNA molecule from a transformed yeast colony. This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes, purification may be impossible.This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing. The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast. (1)

1)Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene. This gene codes for orotidine-5¢-phosphate decarboxylase (an enzyme that catalyses one of the steps in the biosynthesis pathway for pyrimidine nucleotides) and is used as a selectable marker in exactly the same way as LEU2. A YIp cannot replicate as a plasmid as it does not contain any parts of the 2mm plasmid, and instead depends for its survival on integration into yeast chromosomal DNA. Integration occurs just as described for a YEp . (1)

2) Yeast replicative plasmids (YRps) are able to multiply as independent plasmids because they carry a chromosomal DNA sequence that includes an origin of replication. Replication origins are known to be located very close to several yeast genes, including one or two which can be used as selectable markers. YRp7 is an example of a replicative plasmid. It is made up of pBR322 plus the yeast gene TRP1. This gene, which is involved in tryptophan biosynthesis, is located adjacent to a chromosomal origin of replication.The yeast DNA fragment present in YRp7 contains both TRP1 and the origin. (1)

(1) "Chapter 7 Cloning Vectors." Gene Cloning. N.p.: n.p., n.d. 135-37. Http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf. 22 Aug. 2005. Web.

        vector/sequence for secretion? Steven

        growth media?

                lysed pyruvate or uracil -Guy                

Figure 7.2 Using the LEU2

gene as a selectable marker in a yeast cloning experiment.

        

Figure 7.4 Cloning with an E.

coli–yeast shuttle vector such as

YEp13.

http://www.blackwellpublishing.com/genecloning/pdfs/chapter7.pdf

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