@@ -105,23 +105,25 @@ export function Engineering() {
<p>We ultimately chose to use PEA as the sensing marker for our engineered bacteria. </p>
<h3>Cycle 2: Construction of the Sensing Module</h3>
<h4>Design & Build</h4>
<p>1</p>
<p>To specifically respond to PEA, we designed a TynA-FeaR-PTynA inducible system. In Escherichia coli, TynA is a monoamine oxidase that can oxidize aromatic amines such as PEA to the corresponding aldehyde, PAG. FeaR is a transcription factor, which was shown to activate PtynA in the presence of aldehydes. Therefore, we designed a plasmid that constantly express TynA and FeaR and another plasmid with the inducibel promoter PTynA and downstream gene to be activated. </p>
<p>However, TynA can oxidize various kinds of aromatic amines and lack specificity to PEA. Through literature reading, we learned that Rottinghaus et al found the mutant TynA-G494S and FeaR-A81L showed a more specific response to PEA and PAG[6]. Therefore, we constructed the plasmid Pcon-FeaR+Pcon-TynA with the two mutants and a plasmid with inducible promoter PTynA and a reporter gene GFP (Figure 4).</p>
<h4>Test</h4>
<p>1</p>
<p>We planned to co-transformed EcN with two plasmids via electroporation (Protocol-1). However, colony PCR suggested we only transformed successfully with plasmid Pcon-feaR-PcontynA and failed in transforming plasmid PTynA-GFP. We tried several times but all came with negative results (Figure 5).</p>
<h4>Learn</h4>
<p>Our experiment group members analyzed reasons carefully and searched for chemical transformation protocol in EcN (Protocol-2). Fortunately, colony PCR showed successful construction of our engineered EcN (Figure 6) and it was verified by DNA sequencing.</p>
<h3>Cycle 3: Optimizing inducing condition of the Sensing Module</h3>
<h4>Design & Build</h4>
<p>1</p>
<p>After transformation of two plasmids in EcN, we tried to validate the PEA-sensing GFP reporting system with different concentrations of PEA (0, 5, 25, 50 and 100 ng/ml) and explored the optimized inducing concentration of PEA.</p>
<h4>Test</h4>
<p>1</p>
<p>After co-culture with different concentraions of PEA for different time (0,4,8,12 and 24h), the fluorescence was measured on microplate reader by excitation at 410 nm and detection of emission at 500 nm. OD600 (absorbance of 600nm) was also measured on microplate reader for normalization.</p>
<h4>Learn</h4>
<p>1</p>
<p>1</p>
<p>Results showed a significant increase in fluorescent intensity along with the increased level of PEA concentration. 100 ng/ml PEA presented the best capability of induction, and showed significant difference compared with other concentrations of PEA starting from 12 hours.</p>
<p>Therefore, the design of sensing module, which detects the rising level of HE risk factor PEA and induces the expression of gene downstream, is feasible. The opmized concentration of PEA can be set at 100 ng/ml. </p>
