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Revision as of 22:42, 17 October 2014

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. It is demonstrated that limited by coating capacity, M13 phage vector can hold a 1500bp insertion at most, and 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 6407b, where 11 individual genes are categorized into three classes, which regulate its replication, coat packaging, and budding respectively.

<a class="fancybox" rel="group" href=""><img src="" style="width:450px; heigth:auto;margin-left:125x" alt="" /></a>

To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary (Figure above). 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 pII and pV. 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 pll 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 Rephelication. The displaced positive strand is recircularized by pll and converted to RF DNA. Later, when sufficient pV has accumulated, pV dimers coat the single strands, earmarking them for assembly. Besides, pI, pIV and pVIII also play important roles in phage enveloping and release.

Experiment Task

In Bacterial Two-hybrid system, the downstream gene which supports the lifecycle of M13 bacteriophage can only be expressed when prey and bait are strongly interacted.. To achieve this process, we need to obtain defective M13 strain and rescue plasmid, and the defective M13 strain that loses breeding ability should be tested whether it can regain its breeding ability with the help of exogenous core gene.

So we modified the M13 bacteriophage vector in the following aspects.

-select an insert site and insert the target gene.

-knock out a core gene in order to prevent the defective vector from breeding itself.

-test whether the vector above can breed with exogenous core gene expressed.

We hacked into life cycle of M13 bacteriophage to build our IgEM

1. A protein coding sequence that we intend to evolve would be integrated into genome of M13 phage, so that the gene can evolve while M13 phage replicates.

2. Delete one crucial gene of the M13 phage, so that it can complete its life cycle only there is a certain gene to compensate its deficient.

3. Introduce mutagenesis module into the host, so that we can generate a library of target protein coding sequence in IgEM.

4. Use B2H system to compensate deficient M13 phage.

5. Use RNAT to control translation.