Difference between revisions of "Team:Technion Israel/Project/Expression"
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<p>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).</p> | <p>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).</p> | ||
| − | <h3> | + | <h3>Activity Check</h3> |
| − | <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 will be surrounded by a mixture of molecules, such as those originated from lysed cells. </p> |
| − | <p><i> | + | <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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<p><i>B.subtilis</i> has also the ability to secrete various proteins. </p> | <p><i>B.subtilis</i> has also the ability to secrete various proteins. </p> | ||
<p>For those reasons, we chose <i>B.subtilis</i> as our chassis organism for excreting the 3α-HSD enzyme of interest.</p> | <p>For those reasons, we chose <i>B.subtilis</i> as our chassis organism for excreting the 3α-HSD enzyme of interest.</p> | ||
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<h2>Expression</h2> | <h2>Expression</h2> | ||
| − | <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. </p> |
| − | + | <img class= "img_center" src= "https://static.igem.org/mediawiki/2015/6/62/Technion_Israel_2015_project_bacillus_pdr111.png" alt="pT7 with 3a-HSD"/></br> | |
| − | + | <p> The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (below).</p> | |
| − | + | <img class= "img_center" src= "https://static.igem.org/mediawiki/2015/e/ee/Technion_Israel_2015_project_bacillus_circuit-gene.png" alt="SDS-page-3a-HSD" /> | |
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| − | <img class= "img_center" src= "https://static.igem.org/mediawiki/2015/e/ee/Technion_Israel_2015_project_bacillus_circuit-gene.png" alt="3a-HSD | + | |
| − | <h2> | + | <h2>Activity Assay</h2> |
| − | <p> | + | <p>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.</p> |
| − | <p> | + | <p>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.</p> |
| − | <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> |
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| − | + | <p> Click here to see our results.</p> | |
| − | <p>Click here to see our results.</p> | + | |
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Revision as of 14:15, 17 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 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.
B.subtilis has also the ability to secrete various proteins.
For those reasons, we chose B.subtilis as our chassis organism for excreting the 3α-HSD enzyme of interest.
In our lab, we used strain PY79 wild type which we received from Dr. Avigdor Eldar’s lab at Tel Aviv University.
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.
The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (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.