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

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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.
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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.
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<h2>Life Cycle of M13 bacteriophage</h2>
<h2>Life Cycle of M13 bacteriophage</h2>
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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.
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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].
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/5/57/SYSU-China_Project-Design-M13_life_cycle.jpg"><img src="https://static.igem.org/mediawiki/2014/5/57/SYSU-China_Project-Design-M13_life_cycle.jpg" style="width:450px; heigth:auto;margin-left:125x" alt="" /></a>
 
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/5/57/SYSU-China_Project-Design-M13_life_cycle.jpg"><img src="https://static.igem.org/mediawiki/2014/5/57/SYSU-China_Project-Design-M13_life_cycle.jpg" style="width:450px; heigth:auto;margin-left:125px" alt="" /></a><br/>
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Figure 1. <b>Life cycle of Enterobacteria phage M13 in host cell.</b> Mainly shows the replication of viral DNA. Thin thread represents single strand DNA, while bold thread represent double strand DNA.
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To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary (Figure above).  
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To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary (Fig. 1).  
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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.
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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.
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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.
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<h2>Experiment Task</h2>
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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].
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<h2>How do we modify the M13 bacteriophage?</h2>
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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.
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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.
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So we modified the M13 bacteriophage vector in the following aspects. </p>
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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.
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Next step, integration.
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-select an insert site and insert the target gene. </p>
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After acquiring the modified M13 vector, to accomplish IgEM integration, we would
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-knock out a core gene in order to prevent the defective vector from breeding itself. </p>
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-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.
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-test whether the vector above can breed with exogenous core gene expressed.</p>
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<h2>We hacked into life cycle of M13 bacteriophage to build our IgEM</h2>
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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.
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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.
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-<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>
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3. Introduce mutagenesis module into the host, so that we can generate a library of target protein coding sequence in IgEM.
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-<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/B2H">Employ B2H system to compensate deficient M13 phage.</a>
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4. Use B2H system to compensate deficient M13 phage.
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-<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/RNAT">Use RNAT to control M13 vector's translation.</a>
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Reference
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5. Use RNAT to control translation.
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[1]Calendar, R. The Bacteriophages (Oxford Univ. Press, 2006) <br />
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[2]van Wezenbeek PM, et al., Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd, Gene 1980<br />
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[3]Russel M, Moving through the membrane with filamentous phages, Trends Microbiol. 1995 Jun;3(6):223-8.
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Latest revision as of 02:32, 18 October 2014

Contents

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].


<a class="fancybox" rel="group" href="SYSU-China_Project-Design-M13_life_cycle.jpg"><img src="SYSU-China_Project-Design-M13_life_cycle.jpg" style="width:450px; heigth:auto;margin-left:125px" alt="" /></a>

<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.






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