Team:Edinburgh/Phage Reactors 1.0

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Revision as of 10:26, 23 June 2011

Bacteria makes phage; BioBrick in bacteria codes for phage capsid proteins; these do something useful like degrade cellulose

There exist situations where:

• several enzymes are needed to produce the desired product
• these enzymes work synergistically
• these enzymes must work outside the cell

This proposal allows for the construction of scaffolds containing several enzymes, as well as ensuring that this complex is exported from the cell. This is accomplished by using phage as the scaffold, and attaching enzymes to it by protein fusion.

There are probably many uses of an external reaction scaffold, so this technique is fairly general and could hopefully be used for many purposes, just by swapping in the correct BioBricks. One example is a [http://en.wikipedia.org/wiki/Cellulosome cellulosome], which is a complex of cellulolytic enzymes that can be used to turn cellulose (tough plant material) into sugar, which can then be fermented into fuel. This is something Chris is quite expert on, so could be a good fit for the lab.

Technical details

The project would probably use phage M13, which is non-lytic (does not kill the bacteria). The major coat protein (p8 aka pVIII) already exists in the Registry as <partinfo>BBa_M13008</partinfo>, although I don't think DNA is available. The entire genome is known, of course, so primers can be designed. Phage itself can be ordered if needed, e.g. [http://www.neb.com/nebecomm/products/productN0315.asp here].

• pVIII forward primer: atg aaa aag tct tta gtc ctc
• pVIII reverse primer: tca gct tgc ttt cga ggt gaa

Ideally, we wouldn't have to work with infectious phage at all. Rather, a phagemid could be designed (or maybe it exists already) that lacks a viable p3 protein and so is non-infectious (so claim [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1221525/pdf/11104694.pdf Cebe and Geiser, 2000]). The phagemid that we can order above carries a kanamycin resistance marker so it will be maintained in cells in the presence of kanamycin.

(A phagemid is a plasmid that contains both a plasmid-type origin of replication, but also a phage-type rolling circle origin so it can form single stranded DNA which is packaged into the phage capsid.)

Interestingly, MIT 2010 claim that if p3 is deleted, very long phage particles will be formed and tethered to the membrane; this may be acceptable.

Proteins can be fused to p8 at its amino terminal (i.e. 5' end in the DNA), according to [http://www.utoronto.ca/sidhulab/pdf/08.pdf Weiss and Sidhu, 2000].

To attach several different proteins to the resulting phage, different fusions can be created and all of them expressed on a plasmid. It may be simplest if this is a different plasmid from the phagemid, though plasmid compatibility will have to be kept in mind.

Chris notes that the presence of repeated sequences on a plasmid can lead to genetic instability, but this will not be a problem in lab strains, which lack an important recombination enzyme. As for the use of this technology in industry, it will be possible to overcome this problem simply by synthesising coding sequences with as many altered (but synonymous) codons as possible. The (non-iGEM?) group [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=course&group=MIT%2020.109%20Spring07 MIT 20.109 Spring07] seem to have thought along these lines, e.g. compare <partinfo>BBa_M13008</partinfo> with <partinfo>BBa_M31281</partinfo>.

(An alternative is just to attach different enzymes to different coat proteins; p3 aka pIII is known to work, maybe one of the other coat proteins as well...)

As proof of concept (i.e. something we can accomplish in a short time) it will suffice to get one protein attached to the phage, as long as we can assay for it being exported from the cell. Better would be to get two enzymes attached, which are both required to produce some assayable product.

A modelling component

This project has a natural modelling component. Cellulosomes contain several different enzymes that assist in the conversion of cellolose into sugar. It would be good to optimise what the ratio of these various enzymes should be. Modelling will be needed for investigation of other possible phage reactors as well.

References

• Cebe R, Geiser M (2000) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1221525/pdf/11104694.pdf Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology]. Biochemical Journal 352: 841-849.

• Weiss GA, Sidhu SS (2000) [http://www.utoronto.ca/sidhulab/pdf/08.pdf Design and evolution of artificial M13 coat proteins]. Journal of Molecular Biology 300: 213-219 (doi: 10.1006/jmbi.2000.3845).

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