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Team:SDU-Denmark/Tour51
From 2013.igem.org
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<h4>pSB1C3-Plac-dxs <span class="specialWord">(E. coli)</span></h4> | <h4>pSB1C3-Plac-dxs <span class="specialWord">(E. coli)</span></h4> | ||
| - | The <span class="specialWord">dxs</span> from <span class="specialWord">E. coli</span> was already present in parts registry (<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K118000">BBa_K118000</a>), which simplified our aim to increase expression of the gene. However, the middle part of the brick was unsequenced, so this was an obvious focal point. The full sequence has been entered into part registry. User cloning was successfully used to clone Plac together with <span class="specialWord">E.coli dxs</span>. The picture shows the colony PCR result for the cloning and subsequent transformation. The gel picture shows the expected length around 2300 bp including verification primers. | + | The <span class="specialWord">dxs</span> from <span class="specialWord">E. coli</span> was already present in parts registry (<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K118000">BBa_K118000</a>), which simplified our aim to increase expression of the gene. However, the middle part of the brick was unsequenced, so this was an obvious focal point. The full sequence has been entered into part registry. User cloning was successfully used to clone Plac together with <span class="specialWord">E.coli dxs</span>. The picture shows the colony PCR result for the cloning and subsequent transformation. The gel picture shows the expected length around 2300 bp including verification primers (Fig.1). |
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<h4>pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span></h4> | <h4>pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span></h4> | ||
| - | The literature suggested that <span class="specialWord">dxs</span> from <span class="specialWord">B. subtilis</span> performed better than its <span class="specialWord">E. coli</span> counterpart. Therefore, a colony PCR was performed on the <span class="specialWord">B. subtilis</span> strain 168. USER cloning was used to produce the construct pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span>. Our tests of the construct showed unfortunately two restriction sites within the coding sequence. | + | The literature suggested that <span class="specialWord">dxs</span> from <span class="specialWord">B. subtilis</span> performed better than its <span class="specialWord">E. coli</span> counterpart. Therefore, a colony PCR was performed on the <span class="specialWord">B. subtilis</span> strain 168. USER cloning was used to produce the construct pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span>. Our tests of the construct showed unfortunately two restriction sites within the coding sequence (Fig. 2). |
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| + | Site directed mutagenesis was performed to remove the restriction sites, which ensured that the gene complied with iGEM standards. The gel picture shows the expected length around 2000 bp (Fig. 3). | ||
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| + | </p> | ||
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| + | Then, our focus moved to expression control: Subsequent testing proved that the inherent amount of LacI (the repressor of the lactose promoter) in MG1655 was insufficient in the presence of our construct in a high-copy plasmid (see characterization). Therefore, LacI with an LVA tag was taken from parts registry (<a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>) and cloned into our construct with its own promoter and terminator. pSB1C3-Pcon-LacI:LVA-term-Plac-dxs(sub) was born. The gel-picture shows the result for the test digestion. Linearized plasmid has the expected length around 5500 bp, and the EcoRI and PstI cut plasmid has expected lengths around 2000 bp and 3500 bp (Fig. 4). | ||
</p> | </p> | ||
Revision as of 23:12, 3 October 2013
Cloning
The beauty of creation
To clarify the progression of our cloning, the results will not be presented chronologically, but rather in a logical manner, starting with the simplest constructs. This is not an exhaustive list, but merely a presentation to clarify our work and give insight into our thoughts. Please consult our submitted parts-page (dig deeper), as well as our comprehensive protocols to gain full insight into our work.
pSB1C3-Plac-dxs (E. coli)
The dxs from E. coli was already present in parts registry (BBa_K118000), which simplified our aim to increase expression of the gene. However, the middle part of the brick was unsequenced, so this was an obvious focal point. The full sequence has been entered into part registry. User cloning was successfully used to clone Plac together with E.coli dxs. The picture shows the colony PCR result for the cloning and subsequent transformation. The gel picture shows the expected length around 2300 bp including verification primers (Fig.1).
pSB1C3-Plac-dxs (B. subtilis)
The literature suggested that dxs from B. subtilis performed better than its E. coli counterpart. Therefore, a colony PCR was performed on the B. subtilis strain 168. USER cloning was used to produce the construct pSB1C3-Plac-dxs (B. subtilis). Our tests of the construct showed unfortunately two restriction sites within the coding sequence (Fig. 2).Site directed mutagenesis was performed to remove the restriction sites, which ensured that the gene complied with iGEM standards. The gel picture shows the expected length around 2000 bp (Fig. 3).
Then, our focus moved to expression control: Subsequent testing proved that the inherent amount of LacI (the repressor of the lactose promoter) in MG1655 was insufficient in the presence of our construct in a high-copy plasmid (see characterization). Therefore, LacI with an LVA tag was taken from parts registry (BBa_C0012) and cloned into our construct with its own promoter and terminator. pSB1C3-Pcon-LacI:LVA-term-Plac-dxs(sub) was born. The gel-picture shows the result for the test digestion. Linearized plasmid has the expected length around 5500 bp, and the EcoRI and PstI cut plasmid has expected lengths around 2000 bp and 3500 bp (Fig. 4).
pSB1C3-Para-HRT2
Reporter systems
A list of our primers
