Line 58: | Line 58: | ||
<p>We constructed the Evaluation Vector (EV) to quickly screen for the secretion of a protein of interest as a composite part containing a specifically/specially designed MCS and transformation success indicators based on the pBS1C backbone <a target="_blank" href ="https://www.ncbi.nlm.nih.gov/pubmed/24295448">[1]</a> (Figure 3). Additionally, this vector can be applied for the expression of any other fusion protein of interest regulated by a promoter of your choice. For more details on the application check out our <a target="_blank" href="https://2017.igem.org/Team:TU_Dresden/Measurement">Signal Peptide Toolbox</a> and <a target="_blank" href= "https://2017.igem.org/Team:TU_Dresden/Project/Secretion">Secretion project</a>.</p> | <p>We constructed the Evaluation Vector (EV) to quickly screen for the secretion of a protein of interest as a composite part containing a specifically/specially designed MCS and transformation success indicators based on the pBS1C backbone <a target="_blank" href ="https://www.ncbi.nlm.nih.gov/pubmed/24295448">[1]</a> (Figure 3). Additionally, this vector can be applied for the expression of any other fusion protein of interest regulated by a promoter of your choice. For more details on the application check out our <a target="_blank" href="https://2017.igem.org/Team:TU_Dresden/Measurement">Signal Peptide Toolbox</a> and <a target="_blank" href= "https://2017.igem.org/Team:TU_Dresden/Project/Secretion">Secretion project</a>.</p> | ||
<p>We provide the MCS as a part stored in the pSB1C3 backbone (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2273107">BioBrick BBa_K2273107</a>).</p> | <p>We provide the MCS as a part stored in the pSB1C3 backbone (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2273107">BioBrick BBa_K2273107</a>).</p> | ||
+ | |||
+ | <figure> | ||
+ | <figure> | ||
+ | <figure class="makeresponsive floatleft" style="width: 33%;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/4d/EvaluationVectorCloning.png" alt="A scheme explaining the cloning of the Evaluation Vector."> | ||
+ | <figcaption><b>Figure 2: Cloning scheme of the Evaluation Vector.</b> The detailed cloning workflow which led to the finished Evaluation Vector construct with the pBS1C backbone.</figcaption></figure> | ||
+ | <figure> | ||
+ | <figure class="makeresponsive floatleft" style="width: 33%;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/4d/EvaluationVectorCloning.png" alt="A scheme explaining the cloning of the Evaluation Vector."> | ||
+ | <figcaption><b>Figure 2: Cloning scheme of the Evaluation Vector.</b> The detailed cloning workflow which led to the finished Evaluation Vector construct with the pBS1C backbone.</figcaption></figure></figure> | ||
+ | |||
<p>As stated above in the Background section of the EV, we aimed for an easy cloning and screening procedure in our cloning host <i>Escherichia coli</i>. To accomplish that, we chose to set the construct RFPsyn2 as placeholder for the N-terminally fused protein and the gene <i>lacZα</i> for the C-terminally fused protein, respectively. Therefore, the blue color of <i>lacZα</i> carrying colonies and thereby X-Gal degrading colonies masks the red color of the RFPsyn2 on X-Gal containing agar plates. However, on not X-Gal containing agar plates, the red color of the RFPsyn2 will be visible. <i> E. coli</i> colonies carrying neither <i>lacZα</i> nor RPFsyn2 will stay whitish as common <i> E. coli</i> colonies (Figure 4). By applying this setup, successfully transformed <i>E. coli</i> colonies can be identified easily, as stated below in the standard operating procedure (SOP) protocol.</p> | <p>As stated above in the Background section of the EV, we aimed for an easy cloning and screening procedure in our cloning host <i>Escherichia coli</i>. To accomplish that, we chose to set the construct RFPsyn2 as placeholder for the N-terminally fused protein and the gene <i>lacZα</i> for the C-terminally fused protein, respectively. Therefore, the blue color of <i>lacZα</i> carrying colonies and thereby X-Gal degrading colonies masks the red color of the RFPsyn2 on X-Gal containing agar plates. However, on not X-Gal containing agar plates, the red color of the RFPsyn2 will be visible. <i> E. coli</i> colonies carrying neither <i>lacZα</i> nor RPFsyn2 will stay whitish as common <i> E. coli</i> colonies (Figure 4). By applying this setup, successfully transformed <i>E. coli</i> colonies can be identified easily, as stated below in the standard operating procedure (SOP) protocol.</p> | ||
<p>Based on our design, we established the following SOP protocol for cloning with the EV.</p> | <p>Based on our design, we established the following SOP protocol for cloning with the EV.</p> |
Revision as of 13:40, 25 October 2017