Team:Wageningen UR/project/low copy

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<h1>Looking for low copy number plasmids</h1>
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<h1>Looking for low copy number plasmids</h1>
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<h2>Overview and Approach</h2>
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<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 origins 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 and pSB3 (p15A Ori), pSB4 (rep101, repA Ori) and pSB6 (pMB1 Ori) which are described as low-medium copy number plasmids. <br/>At the beginning of the project we used pS1K3 and pSB6A3 and to our surprise the plasmid concentrations of the miniprepped 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. The comparison was done by growing 10 mL <i>E. coli DH5alpha</i> in LB medium to an OD600 of 0.6. Samples were diluted to obtain the exact OD and then equal volumes were miniprepped. Yields were then determined by Nanodrop, as well as running the samples in an agarose gel. All this was done in triplicates, except for the high copy number plasmid pSB1K3 that was preformed in duplo.</p>
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<p>Two sets of T-A combinations, <b>Kid-Kis</b> and <b>Zeta-Epsilon</b> which have been studied for biosafety purposes before, were selected for this project [2]. Both are type II T-A systems, meaning that antitoxin protein prevents the toxin protein from functioning by binding. </p>
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<h2>Results</h2>
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<p> The performed experiments show that of 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 the rest. Quit surprising, pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid.</p>
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<figcaption> Figure 1. Mode of action of the double interdependent plasmid system that avoids horizontal gene transfer between BananaGuard (green bacteria) and native bacteria in the soil (yellow bacteria). In case one of the plasmids would be transfer to soil bacteria, it would die due to expression of one of the toxins. </figcaption>
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<img src="https://static.igem.org/mediawiki/2014/e/e4/Wageningen_UR_copynumbers.png" width="95%">
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<figcaption> Figure 1. Copy number plasmid of the different plasmids of the iGEM registry. a) Representation of the miniprepped plasmid concentrations obtained from the different copy number plasmids. b) Gel electrophoresis of the different copy number plasmids.</figcaption>
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<h2>Conclusions</h2>
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<p>These findings are relevant for all iGEM teams from the first day when they will plan their experiments based on the information found in the registry. Low copy number plasmids are a key tool to build biological systems. Teams should therefore be careful in their choice of backbone and make sure the low copy number plasmids used are well characterized. The addition of new or improved low copy number plasmids can therefore be considered a goal for the coming years.</p>
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<p style="float:right"><b>Continue to <a href="https://2014.igem.org/Team:Wageningen_UR/project/model_overview"  class="soft_link">Model >></a>  </b>
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Latest revision as of 00:30, 18 October 2014

Wageningen UR iGEM 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 origins 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 and pSB3 (p15A Ori), pSB4 (rep101, repA Ori) and pSB6 (pMB1 Ori) which are described as low-medium copy number plasmids.
At the beginning of the project we used pS1K3 and pSB6A3 and to our surprise the plasmid concentrations of the miniprepped 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. The comparison was done by growing 10 mL E. coli DH5alpha in LB medium to an OD600 of 0.6. Samples were diluted to obtain the exact OD and then equal volumes were miniprepped. Yields were then determined by Nanodrop, as well as running the samples in an agarose gel. All this was done in triplicates, except for the high copy number plasmid pSB1K3 that was preformed in duplo.


Results

The performed experiments show that of 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 the rest. Quit surprising, pSB6 has a higher copy number plasmid than pSB1, the iGEM standard high copy number plasmid.

Figure 1. Copy number plasmid of the different plasmids of the iGEM registry. a) Representation of the miniprepped plasmid concentrations obtained from the different copy number plasmids. b) Gel electrophoresis of the different copy number plasmids.

Conclusions

These findings are relevant for all iGEM teams from the first day when they will plan their experiments based on the information found in the registry. Low copy number plasmids are a key tool to build biological systems. Teams should therefore be careful in their choice of backbone and make sure the low copy number plasmids used are well characterized. The addition of new or improved low copy number plasmids can therefore be considered a goal for the coming years.


Continue to Model >>

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