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− | <h1 align="center">3-1-1 Adjustment of MazF Expression</h1> | + | <h1 align="center">3-1-1 Adjustment of MazF Expression<br><br></h1> |
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<h3 class="link"><a href="#introduction">1. Introduction</a></h3> | <h3 class="link"><a href="#introduction">1. Introduction</a></h3> | ||
− | <h3 class="link"><a href="#summary">2. Summary of the | + | <h3 class="link"><a href="#summary">2. Summary of the experiment</a></h3> |
− | <h3 class="link"><a href="#results">3. Results | + | <h3 class="link"><a href="#results">3. Results & discussion</a></h3> |
− | + | <h3 class="link"><a href="#methods">4. Materials and methods</a></h3> | |
− | <h3 class="link"><a href="#methods"> | + | <h3 class="link"><a href="#construction"><font size="2.7"> 4-1. Construction</font></a></h3> |
− | <h3 class="link"><a href="#construction"><font size="2.7"> | + | <h3 class="link"><a href="#protocol"><font size="2.7"> 4-2. Assay protocol</font></a></h3> |
− | <h3 class="link"><a href="#protocol"><font size="2.7"> | + | <h3 class="link"><a href="#reference">5. Reference</a></h3> |
− | <h3 class="link"><a href="#reference"> | + | |
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<div id="introduction_contents" class="container_contents"> | <div id="introduction_contents" class="container_contents"> | ||
− | <p class="normal_text">In order to control cell growth as we desire using the <span style ="font-style : italic">mazEF</span> system, it is necessary to adjust the expression level of <span style ="font-style : italic">mazF</span>. It has been reported that MazF has very strong ability to inhibit cell growth and that <span style ="font-style : italic">mazE</span> expression can not recover it when <span style ="font-style : italic">mazF</span> is expressed at high level. Therefore, we here explored the relationship between concentration of the expression inducer for <span style ="font-style : italic">mazF</span> ( | + | <p class="normal_text">In order to control cell growth as we desire using the <span style ="font-style : italic">mazEF</span> system, it is necessary to adjust the expression level of <span style ="font-style : italic">mazF</span>. It has been reported that MazF has very strong ability to inhibit cell growth and that <span style ="font-style : italic">mazE</span> expression can not recover from it when <span style ="font-style : italic">mazF</span> is expressed at high level[1]. Therefore, we here explored the relationship between concentration of the expression inducer for <span style ="font-style : italic">mazF</span> (arabinose in this experiment) and expression level of it; such information is important for operating our final genetic circuits properly. |
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<div id="summary" class="container"> | <div id="summary" class="container"> | ||
<div id="id_header" class="container_header"> | <div id="id_header" class="container_header"> | ||
− | <h2><span>2. Summary of the | + | <h2><span>2. Summary of the experiment</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="summary_contents" class="container_contents"> | <div id="summary_contents" class="container_contents"> | ||
− | <p class="normal_text">A pSB6A1-based plasmid containing both the PBAD | + | <p class="normal_text">A pSB6A1-based plasmid containing both the PBAD ‐ <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">mazF</span> and the Pcon ‐ <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">gfp</span> cassettes was constructed. For control experiments, a pSB6A1-based plasmid containing only the Pcon - <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">gfp</span> cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac ‐ <span style ="font-style : italic">rbs</span> cassette was constructed. These plasmids were co-introduced into <span style ="font-style : italic">E. coli</span> cells of which growth was controlled by the expression of <span style ="font-style : italic">mazF</span>.<br> |
− | + | <p class="normal_text">The plasmid shown in Fig. 3-1-1-2-2 (PBAD ‐ <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">mazF</span> and the Pcon ‐ <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">gfp</span>) has been registered as Best Composite Part (<a href="http://parts.igem.org/Part:BBa_K1949102">BBa_K1949102</a>).<br><br> | |
− | <p class="normal_text"><span style ="font-style : italic">E. coli</span> A : carrying the Pcon | + | <p class="normal_text">Transformants shown below were prepared.<br> |
− | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> A</span> | + | |
+ | <h3 id="fig.3-1-1-2-1"> </h3> | ||
+ | <p class="normal_text"><span style ="font-style : italic">E. coli</span> A: carrying the Pcon ‐ <span style ="font-style : italic">rbs ‐ gfp</span> (pSB6A1) , Plac ‐ <span style ="font-style : italic">rbs</span> (pSB3K3)</p> | ||
+ | <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f2/T--Tokyo_Tech--3-1-2-2-5-2.png" height="150"><br></div> | ||
+ | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-1 </span> Plasmid diagram of <span style ="font-style : italic">E. coli</span> A</span> | ||
</p></div><br> | </p></div><br> | ||
− | <p class="normal_text"><span style ="font-style : italic">E. coli</span> B : carrying the PBAD | + | <h3 id="fig.3-1-1-2-2"> </h3> |
− | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-2 Plasmid diagram of <span style ="font-style : italic">E. coli</span> B</span> | + | <p class="normal_text"><span style ="font-style : italic">E. coli</span> B: carrying the PBAD ‐ <span style ="font-style : italic">rbs ‐ mazF ‐ tt</span> ‐ Pcon ‐ <span style ="font-style : italic">rbs ‐ gfp</span> (pSB6A1) , Plac ‐ <span style ="font-style : italic">rbs</span> (pSB3K3)</p> |
+ | |||
+ | <h3 id="fig.3-1-2-1-3-2"> </h3> | ||
+ | <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--3-1-2-2-5-5.png" height="250"><br></div> | ||
+ | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-2 </span> Plasmid diagram of <span style ="font-style : italic">E. coli</span> B</span> | ||
</p></div><br> | </p></div><br> | ||
+ | |||
+ | <p class="normal_text">To express <span style ="font-style : italic">mazF</span>, arabinose was added so that the final concentration became 0.2, 0.02, 0.002, 0.0002, and 0%. Samples were incubated with vigorous shaking for 24 h at 37℃, and the turbidity and the Relative Fluorescence Units (RFU) of GFP were measured.</p> | ||
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<div id="results" class="container"> | <div id="results" class="container"> | ||
<div id="results_header" class="container_header"> | <div id="results_header" class="container_header"> | ||
− | <h2><span>3. Results</span></h2> | + | <h2><span>3. Results & discussion</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="results_contents" class="container_contents"> | <div id="results_contents" class="container_contents"> | ||
− | <p class="normal_text">It was found that | + | <p class="normal_text">It was found that cell growth of <span style ="font-style : italic">E. coli</span> B was inhibited under arabinose (inducer for <span style ="font-style : italic">mazF</span>) concentrations of over 0.02% (<a href="#fig.3-1-1-3-1">Fig.3-1-1-3-1</a>). Interestingly, at arabinose concentration of 0.2%, RFU of GFP fell markedly, regardless of <span style ="font-style : italic">mazF</span> expression, suggesting that high arabinose concentrations may inhibit <span style ="font-style : italic">gfp</span> expression or prevent GFP from exerting fluorescence. Taken together, it is concluded that the concentration of arabinose should be 0.02%. |
</p><br> | </p><br> | ||
− | <h3 id="fig.1"> </h3> | + | <h3 id="fig.3-1-1-3-1"> </h3> |
− | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6b/T--Tokyo_Tech--Description_Toxin1.png" width="400px"/> | + | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6b/T--Tokyo_Tech--Description_Toxin1.png" width="400px"/></div> |
− | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-3-1 | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-3-1 </span> Relative value of turbidity, RFU of GFP and RFU of GFP / turbidity. <a href="#fig.3-1-1-2-1"><span style ="font-style : italic">E. coli</span> A</a> and <a href="#fig.3-1-1-2-2"><span style ="font-style : italic">E. coli</span> B</a> were cultured in the presence of indicated amounts of arabinose, and turbidity (upper graph), RFU of GFP (middle graph), and RFU / turbidity (lower graph) were measured. Also, the same cells were cultured in the absence of arabinose, and measurements were performed similarly as above. The ratio of the former values to the latter values were calculated. |
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<div id="methods" class="container"> | <div id="methods" class="container"> | ||
<div id="methods_header" class="container_header"> | <div id="methods_header" class="container_header"> | ||
− | <h2><span> | + | <h2><span>4. Materials and methods</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="methods_contents" class="container_contents"> | <div id="methods_contents" class="container_contents"> | ||
<div id="construction"> | <div id="construction"> | ||
<div id="construction_header"> | <div id="construction_header"> | ||
− | <h3><span> | + | <h3><span>4-1. Construction</span></h3> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="construction_contents"> | <div id="construction_contents"> | ||
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<p class="normal_text">-Plasmids<br> | <p class="normal_text">-Plasmids<br> | ||
− | + | <a href="#fig.3-1-1-2-1"><span style ="font-style : italic">E. coli</span> A</a>: Pcon ‐ <span style ="font-style : italic">rbs</span> ‐ <span style ="font-style : italic">gfp</span> (pSB6A1) + Plac ‐ <span style ="font-style : italic">rbs</span> (pSB3K3)<br> | |
− | + | <a href="#fig.3-1-1-2-2"><span style ="font-style : italic">E. coli</span> B</a>: PBAD ‐ <span style ="font-style : italic">rbs</span> ‐ <span style="font-style : italic">mazF</span> ‐ <span style ="font-style : italic">tt</span> ‐ Pcon ‐ <span style ="font-style : italic">rbs</span> ‐ <span style="font-style : italic">gfp</span> (pSB6A1) + Plac ‐ <span style ="font-style : italic">rbs</span> (pSB3K3)</p><br> | |
− | <p class="normal_text">PBAD ( | + | <p class="normal_text">PBAD (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) , Plac (<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>) , Pcon (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>) , <span style ="font-style : italic">gfp</span> (<a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a>) ,<br> |
+ | <span style ="font-style : italic">rbs</span> (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) , <span style ="font-style : italic">mazF</span> (<a href="http://parts.igem.org/Part:BBa_K1096002">BBa_K1096002</a>) , <span style ="font-style : italic">tt</span> (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>) | ||
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<div id="protocol"> | <div id="protocol"> | ||
<div id="protocol_header"> | <div id="protocol_header"> | ||
− | <h3><span> | + | <h3><span>4-2. Assay protocol</span></h3> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="methods_contents"> | <div id="methods_contents"> | ||
<p class="normal_text">Pre-culture<br><br> | <p class="normal_text">Pre-culture<br><br> | ||
− | + | 1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br><br> | |
− | + | 2. Incubate with vigorous shaking for 12 h.<br><br><br> | |
<p class="normal_text">Incubation and Assay<br><br> | <p class="normal_text">Incubation and Assay<br><br> | ||
− | + | 1. Measure the turbidity of the pre-cultures.<br><br> | |
− | + | 2. Dilute the pre-cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.<br><br> | |
− | + | 3. Incubate with vigorous shaking so that the turbidity becomes 0.03.<br><br> | |
− | + | 4. Add arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002% 0.0002% and 0%.<br><br> | |
− | + | 5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP. | |
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<div id="reference" class="container"> | <div id="reference" class="container"> | ||
<div id="reference_header" class="container_header"> | <div id="reference_header" class="container_header"> | ||
− | <h2><span> | + | <h2><span>5. Reference</span></h2> |
</div><!-- /_header --> | </div><!-- /_header --> | ||
<div id="reference_contents" class="container_contents"> | <div id="reference_contents" class="container_contents"> | ||
− | <p class="normal_text">1 | + | <h3 id="reference3-1-1-6-1"> </h3> |
+ | <p class="normal_text">[1] Hazan, R., B. Sat, and H. Engelberg-Kulka. <span style ="font-style : italic">E. coli</span> <span style ="font-style : italic">mazEF</span> mediated cell death is triggered by various stressful conditions. J. Bacteriol. 2004 Dec;186(24):8295-8300. | ||
</p> | </p> | ||
</div><!-- /reference_contents --> | </div><!-- /reference_contents --> | ||
</div><!-- /reference --> | </div><!-- /reference --> | ||
+ | |||
+ | <div id="next_page" class="container container_bottom"> | ||
+ | <div id="next_page_contents" class="container_contents"> | ||
+ | <p class="normal_text">Jump to <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/mazEF_System_Assay"><span style="font-style:italic;">mazEF</span> System Assay</a> page.</p> | ||
+ | </div><!-- /next_page_contents --> | ||
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Latest revision as of 11:54, 6 November 2016
![](https://static.igem.org/mediawiki/2016/f/f6/T--Tokyo_Tech--goTop.png)
3-1-1 Adjustment of MazF Expression
Contents
1. Introduction
In order to control cell growth as we desire using the mazEF system, it is necessary to adjust the expression level of mazF. It has been reported that MazF has very strong ability to inhibit cell growth and that mazE expression can not recover from it when mazF is expressed at high level[1]. Therefore, we here explored the relationship between concentration of the expression inducer for mazF (arabinose in this experiment) and expression level of it; such information is important for operating our final genetic circuits properly.
2. Summary of the experiment
A pSB6A1-based plasmid containing both the PBAD ‐ rbs ‐ mazF and the Pcon ‐ rbs ‐ gfp cassettes was constructed. For control experiments, a pSB6A1-based plasmid containing only the Pcon - rbs ‐ gfp cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac ‐ rbs cassette was constructed. These plasmids were co-introduced into E. coli cells of which growth was controlled by the expression of mazF.
The plasmid shown in Fig. 3-1-1-2-2 (PBAD ‐ rbs ‐ mazF and the Pcon ‐ rbs ‐ gfp) has been registered as Best Composite Part (BBa_K1949102).
Transformants shown below were prepared.
E. coli A: carrying the Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)
![](https://static.igem.org/mediawiki/2016/f/f2/T--Tokyo_Tech--3-1-2-2-5-2.png)
Fig. 3-1-1-2-1 Plasmid diagram of E. coli A
E. coli B: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)
![](https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--3-1-2-2-5-5.png)
Fig. 3-1-1-2-2 Plasmid diagram of E. coli B
To express mazF, arabinose was added so that the final concentration became 0.2, 0.02, 0.002, 0.0002, and 0%. Samples were incubated with vigorous shaking for 24 h at 37℃, and the turbidity and the Relative Fluorescence Units (RFU) of GFP were measured.
3. Results & discussion
It was found that cell growth of E. coli B was inhibited under arabinose (inducer for mazF) concentrations of over 0.02% (Fig.3-1-1-3-1). Interestingly, at arabinose concentration of 0.2%, RFU of GFP fell markedly, regardless of mazF expression, suggesting that high arabinose concentrations may inhibit gfp expression or prevent GFP from exerting fluorescence. Taken together, it is concluded that the concentration of arabinose should be 0.02%.
![](https://static.igem.org/mediawiki/2016/6/6b/T--Tokyo_Tech--Description_Toxin1.png)
Fig. 3-1-1-3-1 Relative value of turbidity, RFU of GFP and RFU of GFP / turbidity. E. coli A and E. coli B were cultured in the presence of indicated amounts of arabinose, and turbidity (upper graph), RFU of GFP (middle graph), and RFU / turbidity (lower graph) were measured. Also, the same cells were cultured in the absence of arabinose, and measurements were performed similarly as above. The ratio of the former values to the latter values were calculated.
4. Materials and methods
4-1. Construction
-Strain
All the samples were XL1-Blue strain.
-Plasmids
E. coli A: Pcon ‐ rbs ‐ gfp (pSB6A1) + Plac ‐ rbs (pSB3K3)
E. coli B: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) + Plac ‐ rbs (pSB3K3)
PBAD (BBa_I0500) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) ,
rbs (BBa_B0034) , mazF (BBa_K1096002) , tt (BBa_B0015)
4-2. Assay protocol
Pre-culture
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).
2. Incubate with vigorous shaking for 12 h.
Incubation and Assay
1. Measure the turbidity of the pre-cultures.
2. Dilute the pre-cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.
3. Incubate with vigorous shaking so that the turbidity becomes 0.03.
4. Add arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002% 0.0002% and 0%.
5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.
5. Reference
[1] Hazan, R., B. Sat, and H. Engelberg-Kulka. E. coli mazEF mediated cell death is triggered by various stressful conditions. J. Bacteriol. 2004 Dec;186(24):8295-8300.
Jump to mazEF System Assay page.