<p> We have re-designed the new genetic U6PT circuit in a way that will allow us to clone the PcPT gene into the same target backbone but after digestion of the mCherry sequence out of the plasmid(fig.11-PcPT genetic circuit)</p>
Step1: Amplifying the PcPT gene with new restriction sites. <br>
...
...
@@ -198,12 +198,12 @@
><th><h4>1st Design</h4></th>
</tr></table>
<p>The two genes of interest: ximD and ximE were designed to be cloned into the same plasmid under the regulations of Lac and Tet respectively as shown in fig.12.</p>
<p> We started with pET_Duet_PcPT_ximD_ximE plasmid. The building of this circuit was done as shown below:<br>
Step1: Reverse PCR to amplify the ximE gene out from the plasmid. This step was done in order to replace the gene of ximE with the same gene but under inducible regulation.
...
...
@@ -220,7 +220,7 @@
Modeling results have shown positive correlation between the concentration of decursinol (the desired product) and the concentration of XimE enzyme (fig.13). </p>
<p>To fulfill a constitutive expression for XimE enzyme, TetR repressor was not cloned into ximD_ximE plasmid as shown in fig.13.</p>
<p><b>To sum up</b>, according to the final design for the second plasmid, ximD is expressed inducibiliy under the Lac operator, however ximE is expressed constitutively to achieve maximal biomanufacturing yield of decursinol. This in accordance to our model that predicts higher decursin to marmesin ratio when the molar fraction of XimE increases. </p>
