Team:Edinburgh/Phage Reactors 2.0

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Revision as of 21:52, 13 July 2011

This is Version 2.0 of the Phage Reactor proposal. Version 1.0 is here.

Overview

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 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 what we want to build: phage with enzymes fused to the protein coat.

Technical notes

The project will 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 DNA is not available. The entire genome is known (e.g. here is the entire M13mp18 genome) so primers can be designed. New England Bioworks claims:

"The major coat protein pVIII is present at ~2700 copies per virion, of which ~10% can be reliably fused to peptides or proteins."

We will infect our E. coli with (more or less) wildtype phage. Our E. coli will have a plasmid coding for a fusion between an enzyme and pVIII. Proteins can be fused to pVIII at its amino terminal (i.e. 5' end in the DNA), according to Weiss and Sidhu, 2000. pVIII has a leader peptide (residues 1-23) that is cleaved out, slightly complicating fusion design.

To attach several different proteins to the "reactor", 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.

We will need to tune expression levels of the fusion versus the wildtype protein.

Preexisting parts with DNA available

pVIII only seems to be available in the composite part <partinfo>BBa_K415151</partinfo>.

pIII is available as <partinfo>BBa_K415138</partinfo> (complete) or <partinfo>BBa_K257001</partinfo> (sans signal sequence).

Six M13 phage attached to a bead. Each phage could have different enzymes attached.

Genetic instability

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 MIT 20.109 Spring07 seem to have thought along these lines, e.g. compare <partinfo>BBa_M13008</partinfo> with <partinfo>BBa_M31281</partinfo>.

Maurice suggests a different approach: make several strains of bacteria each producing phage with just one type of pVIII-fusion. But also make each phage have a pIII-fusion with a protein which could bind some sort of bead. The bead now becomes the complete "reactor". Since each bacteria codes for only one pVIII fusion, there is no repeated sequence problem.

The F plasmid

M13 can only infect E. coli if it has a sex pilus. This is coded for by the F plasmid. However, such plasmids are quickly lost from cultures in a laboratory setting, as they confer a fitness cost. The JM109 strain has proline synthesis genes (proAB) absent from its main chromosome, but present on the F plasmid, and so when it is grown on minimal media, the plasmid will not be lost, as it is needed to keep the bacteria alive.

Problems

The question is how efficiently fusions to pVIII can get onto the phage. There are some dire warnings in the literature:

In our experience, most large proteins display well below one copy per phage particle. - Sidhu et al (2000)
A large 20 kDa protein (human growth hormone, hGH) is not displayed at detectable levels. - Weiss and Sidhu (2000)
The properties of the pIV channel may be one of the factors that limit the size of polypeptides that can be displayed on pVIII. - Karlsson (2004)
As a general rule, the minor coat proteins will display larger proteins more effectively than pVIII. - Kehoe and Kay (2005)
[It is plausible] that a phage containing pVIII with a large peptide may be too large in diameter to pass through the 7-nm pIV exit pore in the outer membrane. - Barbas et al (2001)
The pVIII site, although very popular for peptide phage display, is not suitable for the efficient display of large polypeptides such as antibodies. - Benhar (2001)

However, Maurice seemed quite upbeat about the prospects when we met him. It's definitely the case that large stuff has been displayed, but the question is whether this is some sort of fluke that only occurs (say) once per phage (i.e. see the Sidhu quote above).

There's also the question of why the literature says things like 20% of pVIII can successfully be made as fusions to "proteins" - I suspect that what phage display authors consider "proteins" are quite a bit smaller than what we consider "proteins"...

Testing

As proof of concept (i.e. something we can accomplish in a short time) perhaps it would be sufficient to get just one fusion protein working. We need to prove that the enzyme part is actually getting out of the cell, so we must demonstrate that some substance which cannot enter the cell is nevertheless being degraded.

A fairly easy test would be to use a fusion of amylase to pVIII, and assay for starch degradation, which is very easy. There is no amylase BioBrick (with DNA available) in the Registry, so we'd have to make it.

Example systems

A simple version of the system would work as follows:

E. coli are grown up containing a plasmid coding for a pVIII fusion gene, i.e:
- Promoter-RBS-LeaderSequence-Amylase-(Linker?)-pVIII.
These E. coli are infected with M13.
They create new phage; some of the modified pVIII proteins incorporate into the capsid.

More complex versions would either incorporate more pVIII fusions, or multiple strains all of which have a pIII-fusion to attach to beads.

Plan of action

For the basic system utilising only pVIII we will need:

Fusion-ready BioBricks of
- Promoter and RBS
- Mature pVIII (coding for residues 24-73)
- The pVIII leader signal sequence (coding for residues 1-23: MKKSLVLKASVAVATLVPMLSFA)
- Amylase
- Cellulases (<partinfo>BBa_K392006</partinfo>, <partinfo>BBa_K392007</partinfo>, <partinfo>BBa_K392008</partinfo>)

References

Some of these were only used in Version 1.0.

Barbas CF, et al (2001) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press.

Benhar I (2001) Biotechnological applications of phage and cell display. Biotechnology Advances 19(1): 1-33 (doi: 10.1016/S0734-9750(00)00054-9).

Cebe R, Geiser M (2000) 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.

Pashke M, Höhne W (2005) A twin-arginine translocation (Tat)-mediated phage display system. Gene 350(1): 79-88 (doi: 10.1016/j.gene.2005.02.005).

Rakonjaca J, Model P (1998) Roles of pIII in filamentous phage assembly. Journal of Molecular Biology 282(1): 25-41 (doi: 10.1006/jmbi.1998.2006).

Sidhu SS, Weiss GA, Wells JA (2000) High copy display of large proteins on phage for functional selections. Journal of Molecular Biology 296(2): 487-495 (doi: 10.1006/jmbi.1999.3465).

Thammawong P, Kasinrerk W, Turner RJ, Tayapiwatana C (2006) Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage. Applied Microbiology and Biotechnology 69: 697-703 (doi: 10.1007/s00253-005-0242-0).

Wang KC, Wang X, Zhong P, Luo PP (2010) Adapter-directed display: a modular design for shuttling display on phage surfaces. Journal of Molecular Biology 395(5): 1088-1101 (doi: 10.1016/j.jmb.2009.11.068).

Weiss GA, Sidhu SS (2000) Design and evolution of artificial M13 coat proteins. Journal of Molecular Biology 300: 213-219 (doi: 10.1006/jmbi.2000.3845).

@@ Line 40: / Line 40: @@
 Maurice suggests a different approach: make several strains of bacteria each producing phage with just one type of pVIII-fusion. But also make each phage have a pIII-fusion with a protein which could bind some sort of bead. The bead now becomes the complete "reactor". Since each bacteria codes for only one pVIII fusion, there is no repeated sequence problem.
+===The F plasmid===
+M13 can only infect ''E. coli'' if it has a sex pilus. This is coded for by the F plasmid. However, such plasmids are quickly lost from cultures in a laboratory setting, as they confer a fitness cost. The JM109 strain has proline synthesis genes (proAB) absent from its main chromosome, but present on the F plasmid, and so when it is grown on minimal media, the plasmid will not be lost, as it is needed to keep the bacteria alive.
 ==Problems==