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Team:SYSU-China/file/Project/Result/M13.html
From 2014.igem.org
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<h1>M13</h1> | <h1>M13</h1> | ||
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| + | <p> | ||
| + | Overall, our job is to construct a suitable modified M13 phage vector for IgEM. The ideal M13 vector for our system is the one which is breeding-defective but can be rescued by exogenous gene supplement. So we design a rescue experiment to verify it. The rescue experiment involves in two plasmids. The first one is the modified M13 phage vector, which fails to produce progeny phage. The other one is the rescue plasmid, an inducible gene expression vector being able to exogenously supply the deleted gene of M13 phage vector. Both vectors would be co-transformed into the host and we would check whether phage progeny can be generated after induction. | ||
| + | </p> | ||
| + | <h1> | ||
| + | Modified M13 phage vector construction | ||
| + | </h1> | ||
| + | <p> | ||
| + | Our basic phage vector, named M13KE (Fi. 1A), is derived from the commercial Phage Display system of New England Biolabs. | ||
| + | |||
| + | We modified the M13KE vector in several aspects. | ||
| + | |||
| + | First, kanamycin resistance cassette (BBa_P1003), as a selective marker, has been inserted into LacZ locus for convenience of further selection. Because deleting an essential gene from M13 bacteriophage would lose its infectivity, generating no virion to infect the host. Also, the deficient M13 vector could not be maintained by the bacteria after transformation. This excessive vector is named M13KdZ (K represents the kanamycin resistance, and dZ means the lacZ is defected) (Fi. 1B). | ||
| + | |||
| + | To ensure that this primarily engineered M13 vector has a chance to be rescued after additional modification, we verified M13KdZ’s ability of generating progeny phage using Infection Assay. Because the fully package phage genome contains the kanamycin resistance gene, the host E.coli ER2738 would gain the capability to form colonies on kanamycin plates (Fig. 1D). | ||
| + | |||
| + | Second, delete a core gene in order to prevent the vector from breeding itself. The gene III of M13 phage is partly deleted from the M13KdZ vector as previous described (Dübel S et al., 2001) using PCR. An XhoI restriction site is left inside the residual gene III fragment with some extra bases on the primer (Figure 1C). This infect-defeated phage vector is named M13KdZdpIII (dpIII means the gene III protein is defected). | ||
| + | |||
| + | All vectros above are confirmed by restriction analysis (Fig. 1A,B,C) and sequencing. Though we didn’t fully sequence the whole vector, the changed part of the vector is verified by sequencing result. | ||
| + | </p> | ||
| + | |||
| + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/7/79/SYSU-China_Project-M13-Result-Fig1.jpg"><img src="https://static.igem.org/mediawiki/2014/7/79/SYSU-China_Project-M13-Result-Fig1.jpg" alt="" /></a> | ||
| + | <p> | ||
| + | After all, the gene yet to be evolved should be inserted properly when IgEM is completed established. | ||
| + | </p> | ||
Revision as of 19:46, 17 October 2014
M13
Overall, our job is to construct a suitable modified M13 phage vector for IgEM. The ideal M13 vector for our system is the one which is breeding-defective but can be rescued by exogenous gene supplement. So we design a rescue experiment to verify it. The rescue experiment involves in two plasmids. The first one is the modified M13 phage vector, which fails to produce progeny phage. The other one is the rescue plasmid, an inducible gene expression vector being able to exogenously supply the deleted gene of M13 phage vector. Both vectors would be co-transformed into the host and we would check whether phage progeny can be generated after induction.
Modified M13 phage vector construction
Our basic phage vector, named M13KE (Fi. 1A), is derived from the commercial Phage Display system of New England Biolabs. We modified the M13KE vector in several aspects. First, kanamycin resistance cassette (BBa_P1003), as a selective marker, has been inserted into LacZ locus for convenience of further selection. Because deleting an essential gene from M13 bacteriophage would lose its infectivity, generating no virion to infect the host. Also, the deficient M13 vector could not be maintained by the bacteria after transformation. This excessive vector is named M13KdZ (K represents the kanamycin resistance, and dZ means the lacZ is defected) (Fi. 1B). To ensure that this primarily engineered M13 vector has a chance to be rescued after additional modification, we verified M13KdZ’s ability of generating progeny phage using Infection Assay. Because the fully package phage genome contains the kanamycin resistance gene, the host E.coli ER2738 would gain the capability to form colonies on kanamycin plates (Fig. 1D). Second, delete a core gene in order to prevent the vector from breeding itself. The gene III of M13 phage is partly deleted from the M13KdZ vector as previous described (Dübel S et al., 2001) using PCR. An XhoI restriction site is left inside the residual gene III fragment with some extra bases on the primer (Figure 1C). This infect-defeated phage vector is named M13KdZdpIII (dpIII means the gene III protein is defected). All vectros above are confirmed by restriction analysis (Fig. 1A,B,C) and sequencing. Though we didn’t fully sequence the whole vector, the changed part of the vector is verified by sequencing result.
<a class="fancybox" rel="group" href="
"><img src="" alt="" /></a>
After all, the gene yet to be evolved should be inserted properly when IgEM is completed established.
