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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>
  
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<h3>Why <i>Bacillus subtilis</i>?</h3>
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<h3>Activity Check</h3>
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<p>When considering which type of bacteria to use for our cloning, we researched the natural microbiota of the scalp.  We found that <i>Bacillus subtilis</i> is prevalent on our scalp. <sup><a href="#fn1" id="ref1">1</a></sup></p>
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<p>The scalp is not the optimal environment for engineered bacteria, since the natural microbiome 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>
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<p><i>B.subtilis</i> is a gram-positive, rod-shaped bacterium found on skin, in the digestive tract, in epithelial wounds, on extremities of the human body, in livestock and in soil. <sup><a href="#fn2" id="ref2">2</a></sup></p></p>
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<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>Today, the bacterium is used commercially in skin care products, foods for human consumption, antibiotic substitutes, etc. (2)</p>
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<p><i>B.subtilis</i> has also the ability to secrete various proteins. </p>
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<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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<p>In our lab, we used strain PY79 wild type which we received from Dr. Avigdor Eldar’s lab at Tel Aviv University.</p>
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<h2>Expression</h2>
 
<h2>Expression</h2>
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<p> As stated above, our goal was to express  the 3α-HSD protein, originally from rat liver, and check whether it is able to fold properly and break down DHT.</p>
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<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>
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<p>For this purpose, we cloned 3α-HSD  into <i>B.subtilis</i> under the inducible P<sub>hyper-spank</sub> promoter.</p>
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<img class= "img_center" src= "https://static.igem.org/mediawiki/2015/0/0b/Technion_Israel_2015_project_expression_circuit_3aHSD.png" width="800px" alt="pT7 with 3a-HSD"/></br>
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<p>pDR111 is a shuttle vector for <i>E.coli</i> and <i>B.subtilis</i>. When in <i>E.coli</i>, an ampicillin resistance gene is expressed and when in <i>B.subtilis</i>, a spectinomycin resistance is expressed. The plasmid contains homologous regions with the <i>B. subtilis</i> genome, enabling gene introduction by recombination into the nonessential amyE locus of the chromosomal DNA as a single copy.<sup><a href="#fn3" id="ref3">3</a></sup></p></p>
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<p> The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (see Figure 1 below).</p>
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<p>The plasmid also contains lacI gene for the inducible promoter P<sub>hyper-spank</sub>, making our inserted genes inducible with the addition of IPTG.</p>
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<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>In the lab, we inserted our gene in the multiple cloning site between the NheI and SalI restriction sites, which can be seen in the basic pDR111 plasmid below.</p>
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<p> In order to predict the target gene expression, we cloned mCherry into <i>B.subtilis</i>, since it is easier to track reporter genes. The simple, planned circuit of the gene in the shuttle vector is featured in the image below.</p>
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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 on pDR111" width="100%"/>
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<h2>Secretion</h2>
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<h2>Activity Assay</h2>
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<p>Secretion of the 3α-HSD enzyme was an extremely important component of our project. The secretion would enable the bacteria to live on the consumer’s scalp, as opposed to being lysed in order to recover the enzyme.  This could reduce the production costs and improve product efficiency over time.</p>
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<p>Though it was exciting to see a <b>bold</b> 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>
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<p>As mentioned previously, <i>B. subtilis</i>, as well as some other gram-positive bacteria, has protein secretion mechanisms and regularly secretes proteins such as various proteases.</p>
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<p>Ideally, we would have checked the enzyme activity by tracking the degradation of its substrate, DHT. But, Since DHT is not detectable as is and we didn’t have a labeled form of it, we conducted the activity check by tracking the degradation of its cofactor, NADPH.</p>
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<p>In order to engineer the bacteria to meet our purposes, we fused our target gene coding for 3α-HSD to the signal peptide (SP) for the gene aprE, which encodes to extracellular alkaline-serine protease (subtilisin E), the most abundant protease secreted to the medium in wildtype <i>B. subtilis</i>. <sup><a href="#fn4" id="ref4">4</a></sup></p>
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<p>NADPH can be measured using 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 fluorescence plate reader.</p>
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<p>By doing so, the  3α-HSD  enzyme could be recognized by the secretion system of the protease and could be excreted into the extracellular medium.</p>
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<p> The circuit can be seen below.</p>
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<img clas="img_center" src="https://static.igem.org/mediawiki/2015/9/90/Technion_Israel_2015_project_bacillus_circuit-gene-sp.png" alt="3a-HSD on pDR111 with SP" width="100%"/>
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<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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<p>Click here to see our results.</p>
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<sup id="fn1">1. Roia, F. C.; Vanderwyk, R. W.:Resident Microbial Flora of the Human Scalp and its Relationship to Dandruff. Journal of The Society of Cosmetic Chemists. 1969, 20, 113-134.</sup></br>
 
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<sup id="fn2">2. Gonzales, D. J.; Haste, N. M.; Hollands, A.; Fleming, T. C.; Hamby, M.; Pogliano, K.; Nizet, V.; Dorrestein, P.C.: Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry. Microbiology 2011, 157, 24985-2492.</sup></br>
 
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<sup id="fn3">3. Härtle, B.; Wehrl, W.; Wiegert, T.; Homuth, G.; Schumann, W.: Development of a New Integration Site within the Bacillus subtilis Chromosome and Construction of Compatible Expression Cassettes. Journal of Bacteriology. 2001, 183, 2696-2699.
 
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</sup></br>
 
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<sup id="fn4">4. Antelmann, H.; Tjalsma, H.; Voigt, B.; Ohlmeier, S.; Bron, S.; Van Djil, Jan Maarten.; Hecker, M.: A Proteomic View on Genome-Based Signal Peptide Predictions. Genome Research. 2001, 11, 1484-1502.
 
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Latest revision as of 22:36, 18 September 2015

Team: Technion 2015

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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 microbiome 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.

pT7 with 3a-HSD

The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (see Figure 1 below).

SDS-page-3a-HSD
Figure 1: SDS page results for 3ɑ-HSD

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. But, Since DHT is not detectable as is and we didn’t have a labeled form of it, we conducted the activity check by tracking the degradation of its cofactor, NADPH.

NADPH can be measured using fluorescence: excitation at a wavelength of 340nm and emission at wavelength of 460nm, so an activity experiment was done using fluorescence plate reader.

Click here to see our results.

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