Difference between revisions of "Team:Technion Israel/Project/Expression"
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<h3>Activity Check</h3> | <h3>Activity Check</h3> | ||
| − | <p>The scalp is not the optimal environment for engineered bacteria, since the natural microflora consists of a variety of bacteria in different growth stages. Thus, the secreted 3α-HSD enzyme will be surrounded by a mixture of molecules, such as those originated from lysed cells. </p> | + | <p>The scalp is not the optimal environment for engineered bacteria, since the natural microflora consists of a variety of bacteria in different growth stages. Thus, the secreted 3α-HSD enzyme in or product will be surrounded by a mixture of molecules, such as those originated from lysed cells. </p> |
<p>To simulate these conditions in-vitro, we conducted a qualitative experiment involving cell lysates from <i>E.coli</i> cells over-expressing the 3ɑ-HSD enzyme.</p> | <p>To simulate these conditions in-vitro, we conducted a qualitative experiment involving cell lysates from <i>E.coli</i> cells over-expressing the 3ɑ-HSD enzyme.</p> | ||
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<h2>Expression</h2> | <h2>Expression</h2> | ||
<p> Because it is easy to genetically modify it, <i>E.coli</i> was chosen at first for both cloning and expression of the 3ɑ-HSD gene. We over-expressed the enzyme using the pT7 promoter in the <i>E.coli</i> BL21 strain, which expresses the pT7 polymerase, using the plasmid construct seen below and submitted as <a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a>. A basic part, without the promoter, was submitted as <a href="http://parts.igem.org/Part:BBa_K1674001" target="_blank">BBa_K1674001</a>.</p> | <p> Because it is easy to genetically modify it, <i>E.coli</i> was chosen at first for both cloning and expression of the 3ɑ-HSD gene. We over-expressed the enzyme using the pT7 promoter in the <i>E.coli</i> BL21 strain, which expresses the pT7 polymerase, using the plasmid construct seen below and submitted as <a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a>. A basic part, without the promoter, was submitted as <a href="http://parts.igem.org/Part:BBa_K1674001" target="_blank">BBa_K1674001</a>.</p> | ||
| − | <img class= "img_center" src= "" alt="pT7 with 3a-HSD"/></br> | + | <img class= "img_center" src= "https://static.igem.org/mediawiki/2015/0/0b/Technion_Israel_2015_project_expression_circuit_3aHSD.png" alt="pT7 with 3a-HSD"/></br> |
<p> The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (see Figure 1 below).</p> | <p> The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (see Figure 1 below).</p> | ||
<figure><img class= "img_center" src= "https://static.igem.org/mediawiki/2015/2/2b/Technion_Israel_2015_SDS_3aHSD.png" width="500px" height="400px" alt="SDS-page-3a-HSD" /><figcaption>Figure 1: SDS page results for 3ɑ-HSD</figcaption></figure> | <figure><img class= "img_center" src= "https://static.igem.org/mediawiki/2015/2/2b/Technion_Israel_2015_SDS_3aHSD.png" width="500px" height="400px" alt="SDS-page-3a-HSD" /><figcaption>Figure 1: SDS page results for 3ɑ-HSD</figcaption></figure> | ||
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<p>NADPH has fluorescence- excitation at a wavelength of 340nm and emission at wavelength of 460nm, so an <a href="https://static.igem.org/mediawiki/2015/7/7e/Technion_Israel_2015_protocols_3aHSDactivity.pdf" target="_blank">activity experiment</a> was done using a plate reader.</p> | <p>NADPH has fluorescence- excitation at a wavelength of 340nm and emission at wavelength of 460nm, so an <a href="https://static.igem.org/mediawiki/2015/7/7e/Technion_Israel_2015_protocols_3aHSDactivity.pdf" target="_blank">activity experiment</a> was done using a plate reader.</p> | ||
| − | <p> Click here to see our results.</p> | + | <p> Click <a href="https://2015.igem.org/Team:Technion_Israel/Project/Results#expression_results" target="_blank"> here</a> to see our results.</p> |
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Revision as of 08:31, 18 September 2015
Expression
Introduction
Aim
To express and secrete our target gene, 3α-hydroxysteroid dehydrogenase (3α-HSD), in and from bacteria. To prove activity of the secreted gene in breaking down dihydrotestosterone (DHT).
Activity Check
The scalp is not the optimal environment for engineered bacteria, since the natural microflora consists of a variety of bacteria in different growth stages. Thus, the secreted 3α-HSD enzyme in or product will be surrounded by a mixture of molecules, such as those originated from lysed cells.
To simulate these conditions in-vitro, we conducted a qualitative experiment involving cell lysates from E.coli cells over-expressing the 3ɑ-HSD enzyme.
Expression
Because it is easy to genetically modify it, E.coli was chosen at first for both cloning and expression of the 3ɑ-HSD gene. We over-expressed the enzyme using the pT7 promoter in the E.coli BL21 strain, which expresses the pT7 polymerase, using the plasmid construct seen below and submitted as BBa_K1674002. A basic part, without the promoter, was submitted as BBa_K1674001.
The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (see Figure 1 below).
Activity Assay
Though it was exciting to see a bold band on the SDS-PAGE, it was only the first step towards secreting an active 3ɑ-HSD enzyme. To get this to the next level, we wanted to show enzymatic activity of the enzyme using a simple qualitative assay.
Ideally, we would have checked the enzyme activity by tracking the degradation of its substrate, DHT. Since DHT is not detectable as is and we didn’t have a labeled form of it, we checked the activity by tracking the degradation of its cofactor- NADPH.
NADPH has fluorescence- excitation at a wavelength of 340nm and emission at wavelength of 460nm, so an activity experiment was done using a plate reader.
Click here to see our results.