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Team:Wageningen UR/project/low copy
From 2014.igem.org
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<h2>Overview and Approach</h2> | <h2>Overview and Approach</h2> | ||
| - | <p> Our team faced several problems in finding different copy number plasmids in the list of <a href="http://parts.igem.org/Help:Plasmid_backbones/Nomenclature" class="soft_link">iGEM standard backbones </a> available in the registry. In total the iGEM registry has eight different Origens of replication (Ori) documented, numbered from pSB1 to pSB8, having different copy numbers. For our current interest | + | <p> Our team faced several problems in finding different copy number plasmids in the list of <a href="http://parts.igem.org/Help:Plasmid_backbones/Nomenclature" class="soft_link">iGEM standard backbones </a> available in the registry. In total the iGEM registry has eight different Origens of replication (Ori) documented, numbered from pSB1 to pSB8, having different copy numbers. For our current interest we will focus on pSB1 (Ori modified pMB1 derived from pUC19), the only documented high copy number plasmid, pSB3 (p15A Ori), pSB4 (rep101, repA Ori) and pSB6 (pMB1 Ori), that are described as low-medium copy number plasmids. At the beginning of the project we used pS1K3 and pSB6A3, and by our surprise the plasmid concentrations of the minipreped cells were in the same range, when we expected significantly different concentrations. This is the main reason why we decided to compare the copy number of the different plasmids by growing 10 mL <i>E. coli</i> in LB medium at an OD600 of 0.6, minipreping it and determining its concentration by Nanodrop, as well as, running the samples in an agarose gel. All these was done in triplicates, except for the high copy number plasmid pSB1K3 that was preformed in duplicates. </p> |
| + | <br> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| - | <p> The performed experiments show that pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid | + | <p> The performed experiments show that from the three plasmids with low or low-medium copy number plasmids in the iGEM registry only <b>pSB3K3</b> is a real low copy number plasmid. Both in Figure 1 a) and b), a low plasmid concentration is detected and in the gel the intensity is much lower than in other situation. Importantly, pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid.</p> |
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2014/e/e4/Wageningen_UR_copynumbers.png" width="95%"> | <img src="https://static.igem.org/mediawiki/2014/e/e4/Wageningen_UR_copynumbers.png" width="95%"> | ||
| - | <figcaption> Figure 1. Copy number plasmid of the different plasmids of the iGEM registry. a) Representation of the minipreped concentrations obtained from the different copy number plasmids. b) Gel electrophoresis of the different copy number plasmids. </figcaption> | + | <figcaption> Figure 1. Copy number plasmid of the different plasmids of the iGEM registry. a) Representation of the minipreped plasmid concentrations obtained from the different copy number plasmids. b) Gel electrophoresis of the different copy number plasmids. </figcaption> |
</figure> | </figure> | ||
<br> | <br> | ||
| - | <p>These findings are relevant for iGEM teams from the first day when they will plan their experiments based on the information found in the registy.</p> | + | <p>These findings are relevant for iGEM teams from the first day when they will plan their experiments based on the information found in the registy. Low copy number plasmids are a key tool to build biological systems, therefore, a good registy and characterization would be highly for many iGEM registry users.</p> |
<br> | <br> | ||
Revision as of 23:18, 17 October 2014
Looking for low copy number plasmids
Overview and Approach
Our team faced several problems in finding different copy number plasmids in the list of iGEM standard backbones available in the registry. In total the iGEM registry has eight different Origens of replication (Ori) documented, numbered from pSB1 to pSB8, having different copy numbers. For our current interest we will focus on pSB1 (Ori modified pMB1 derived from pUC19), the only documented high copy number plasmid, pSB3 (p15A Ori), pSB4 (rep101, repA Ori) and pSB6 (pMB1 Ori), that are described as low-medium copy number plasmids. At the beginning of the project we used pS1K3 and pSB6A3, and by our surprise the plasmid concentrations of the minipreped cells were in the same range, when we expected significantly different concentrations. This is the main reason why we decided to compare the copy number of the different plasmids by growing 10 mL E. coli in LB medium at an OD600 of 0.6, minipreping it and determining its concentration by Nanodrop, as well as, running the samples in an agarose gel. All these was done in triplicates, except for the high copy number plasmid pSB1K3 that was preformed in duplicates.
Results
The performed experiments show that from the three plasmids with low or low-medium copy number plasmids in the iGEM registry only pSB3K3 is a real low copy number plasmid. Both in Figure 1 a) and b), a low plasmid concentration is detected and in the gel the intensity is much lower than in other situation. Importantly, pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid.
These findings are relevant for iGEM teams from the first day when they will plan their experiments based on the information found in the registy. Low copy number plasmids are a key tool to build biological systems, therefore, a good registy and characterization would be highly for many iGEM registry users.
