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Team:SYSU-China/file/Project/Design/M13.html
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M13 bacteriophage group | M13 bacteriophage group | ||
Revision as of 14:47, 17 October 2014
M13
M13 bacteriophage group Design Why we chose M13 phage 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 M13 bacteriophage as the gene carrier of the protein we aimed to evolve.
Under laboratory condition, M13 can breed a generation every ten minutes. It is demonstrated that limited by coating capacity, M13 bacteriophage 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.
M13 life cycle（配图） 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.
To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary. 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.
We hacked into M13 life cycle to build our IgEM.(followings with linker) Insert a gene that we want to evolve into M13 genome, so that the gene can evolve while M13 phage replicate. Knock out one of the gene of the M13 phage, so that it can complete its life cycle when there is a certain gene to compensate its deficient. Introduce mutagenesis module to the insertion sequence, so that we can generate a library of target gene in the IgEM. Use B2H system to compensate deficient M13 Use RNAT to control translation.
