From 2014.igem.org

M13·DESIGN

Why do we choose M13 bacteriophage?

Our project aims to provide a method for artificial protein evolution, so the selection of target protein carrier is very important. A suitable vector should have a short proliferation cycle and provide enough capacity for target gene. Thus, based on comparison among different vectors, we decided to use Enterobacteria phage M13 as the gene carrier of the protein we aimed to evolve.

Under laboratory condition, M13 bacteriophage can breed a generation every ten minutes[1]. It is demonstrated that capacity of M13 phage vector is limited, but if part of its genome knocked-out, successful expression of larger proteins can be somewhat expected.

Life Cycle of M13 bacteriophage

Enterobacteria phage M13 is a filamentous bacteriophage composed of protein capsid and a circular single stranded DNA (ssDNA) genome of 6407bp[2], where 11 individual genes are categorized into three classes, which regulate its replication, coat packaging, and budding respectively[4].

<p1> Figure 1. Life cycle of Enterobacteria phage M13 in host cell. Mainly shows the replication of viral DNA. Thin thread represents single strand DNA, while bold thread represent double strand DNA. </p1>

To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary (Fig. 1). The general stages of M13 life cycle comprises: infection, genome replication, assembly of new particles, and then release of the offspring particles from the host.

The genome replication is firmly connected with G2P and G5P. The single-stranded phage DNA that enters the cell is converted to a supercoiled, double-stranded replicative form (RF) by several host enzymes. Phage gene expression ensues, and G2P nicks the viral strand at the positive-strand origin. The 3' end of the nick is extended by DNA polymerase III, single-strand-binding protein and the Replication. The displaced positive strand is re-circularized by pll and converted to RF DNA. Later, when sufficient G5P has accumulated, G5P dimers coat the single strands, earmarking them for assembly. Besides, G1P, G4P and G8P also play important roles in phage enveloping and release[3].

How do we modify the M13 bacteriophage?

In IgEM, the life cycle of M13 bacteriophage could be accomplished only when reporter gene of Bacterial Two-hybrid System is supplied, meaning the prey and bait are strongly interacted. To achieve this process, we need to obtain defective M13 vector, of which breeding ability is abolished. Meanwhile, this vector should re-gain its breeding ability with exogenous gene expression supplement. Thus we modified the M13 bacteriophage vector in the following aspects.

First, delete a core gene to prevent the defective vector from breeding itself. Then, test whether the vector above can breed with exogenous core gene supplied.

Next step, integration.

After acquiring the modified M13 vector, to accomplish IgEM integration, we would

-Integrate a protein coding sequence that we intend to evolve into <https://2014.igem.org/Team:SYSU-China/content.html#Project/Result/M13">defective M13 phage vector</a>, enabling its evolution while M13 phage replicates.

-<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/Mutagenesis">Introduce mutagenesis module into the host, so that we can generate a library of target protein coding sequence in IgEM.</a>

-<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/B2H">Employ B2H system to compensate deficient M13 phage.</a>

-<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/RNAT">Use RNAT to control M13 vector's translation.</a>

Reference

[1]Calendar, R. The Bacteriophages (Oxford Univ. Press, 2006)
[2]van Wezenbeek PM, et al., Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd, Gene 1980
[3]Russel M, Moving through the membrane with filamentous phages, Trends Microbiol. 1995 Jun;3(6):223-8.

Team:SYSU-China/file/Project/Design/M13.html