Difference between revisions of "Team:ZJU-China/Safety"

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<h1> Safety </h1>
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<p>Please visit the <a href="https://2018.igem.org/Safety">Safety Hub</a> to find this year's safety requirements & deadlines, and to learn about safe & responsible research in iGEM.</p>
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<p>On this page of your wiki, you should write about how you are addressing any safety issues in your project. The wiki is a place where you can <strong>go beyond the questions on the safety forms</strong>, and write about whatever safety topics are most interesting in your project. (You do not need to copy your safety forms onto this wiki page.)</p>
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<h3>Safe Project Design</h3>
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<p>Does your project include any safety features? Have you made certain decisions about the design to reduce risks? Write about them here! For example:</p>
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<li>Choosing a non-pathogenic chassis</li>
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<li>Choosing parts that will not harm humans / animals / plants</li>
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<li>Substituting safer materials for dangerous materials in a proof-of-concept experiment</li>
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<li>Including an "induced lethality" or "kill-switch" device</li>
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<h3>Safe Lab Work</h3>
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<p>What safety procedures do you use every day in the lab? Did you perform any unusual experiments, or face any unusual safety issues? Write about them here!</p>
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<p>Did you face any safety problems in sending your DNA parts to the Registry? How did you solve those problems?</p>
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<div class="title">
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<div class="tl">SAFETY&nbsp;</div>
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</div>
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<div class="intro">
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<p>Currently, T7 promoter is one of the most widely used promoters for expression of heterogenous protein in some E.coli strains such as BL21(DE3). Though the wild-type T7 promoter has proven quite effective, in some cases, we need modified T7 promoters with even higher efficiency of protein expression to meet specific demands. Hence, we tried to transform the wild-type T7 promoter to get modified T7 promoters with increased strength . </p>
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</br>
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<p>T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+ 1) and extends from -17 to +6. As reported in some papers, the sequence specificty of T7 promoter is so strong that some point mutations between positions -11 and -7 may make T7 promoter fail to work. Thus, with the help of previous research, we carefully chose the site which would be mutated by PCR. These sites mainly distribute in the range from -4 to +6. The sequences of these modified promoters are shown in the Table below.</p>
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</br>
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</div>
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<hr />
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<!--<div class="element1">
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<h5>Engaging Experts</h5>
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<p>High impact projects with consequences affecting whole humanity should always be discussed with professionals from different scientific backgrounds. To meet this goal we talked to theologists, legal professionals, safety representatives, astrophysicists, and experts in the field of medicine, agriculture, information technology and many more. Check out the professionals who shaped our project!</p>
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</div>-->
 +
<div class="cnt_psg">
 +
<div class="cnt"></div>
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<div class="psg" >
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<span class="psg_ttl">Sequences of Modified Promoters</span>
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<table class="table">
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<thead>
 +
<tr>
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<th><span>Part Number</span></th>
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<th><span>Sequence(-17~+6)</span></th>
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</tr>
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</thead>
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<tbody>
 +
<tr>
 +
<td><span>BBa_R0085(wild type)</span></td>
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<td><span>TAATACGACTCACTATAGGGAGA</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721000</span></td>
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<td><span>TAATACGACTCACTAT<span style="color: steelblue;">C</span><span></span>G</span><span style="color: steelblue;">C</span><span>G</span><span style="color: steelblue;">GAG</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721001</span></td>
 +
<td><span>TAATACGACTCACT</span><span style="color: steelblue;">CC</span><span>AG</span><span style="color: steelblue;">CA</span><span>A</span><span style="color: steelblue;">TC</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721002</span></td>
 +
<td><span>TAATACGACTCACT</span><span style="color: steelblue;">TC</span><span>AG</span><span style="color: steelblue;">C</span><span>GA</span><span style="color: steelblue;">CC</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721003</span></td>
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<td><span>TAATACGACTCAC</span><span style="color: steelblue;">ACG</span><span>AG</span><span style="color: steelblue;">C</span><span>GAGA</span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<span class="psg_ttl">Test</span>
 +
<p>To test the function of mutant promoters, we chose the GFP as our reporter. By assessing the absolute fluorescence units(RFU) and OD600, we can conclude the relative strength of all promoters. When the E.coli BL21(DE3) is cultured at the stage of logarithmic phase, we added IPTG to induce the expression of GFP in strains BL21(DE3) for 4 hours. And the result is shown as Figure and Table below.</p>
 +
<img src="https://static.igem.org/mediawiki/2018/5/54/T--ZJU-China--ip01.png" style="width: 80%;"/>
 +
<h5>Fig.1 Relative Strength of wildtype T7 promoter and mutant promoters</h5>
 +
<table class="table">
 +
<thead><tr>
 +
<th><span>Part Number</span></th>
 +
<th><span>Relative Strength</span></th>
 +
</tr></thead>
 +
<tbody>
 +
<tr>
 +
<td><span>BBa_R0085(wild type)</span></td>
 +
<td><span>1</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721000</span></td>
 +
<td><span>20.99</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721001</span></td>
 +
<td><span>17.75</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721002</span></td>
 +
<td><span>7.63</span></td>
 +
</tr>
 +
<tr>
 +
<td><span>BBa_K2721003</span></td>
 +
<td><span>13.92</span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p></br>As we can see from the figure, our mutant promoters showed largely increased strength compared with wild type T7 promoter. Therefore, our mutant promoters offer users more opportunity to control the expression of protein using T7 promoter and permit higher levels of target protein expression to be obtained.</p>
 +
<span class="psg_ttl">References</span>
 +
<p>[1] Ikeda R A, Ligman C M, Warshamana S, et al. T7 promoter contacts essential for promoter activity in vivo[J]. Nucleic Acids Research, 1992, 20(10): 2517-2524.</p>
 +
<p>[2] Paul S, Stang A, Lennartz K, Tenbusch M, Uberla K. Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution[J]. Nucleic Acids Research, 2013, 41(1):e29.</p>
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Revision as of 12:28, 13 October 2018

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Currently, T7 promoter is one of the most widely used promoters for expression of heterogenous protein in some E.coli strains such as BL21(DE3). Though the wild-type T7 promoter has proven quite effective, in some cases, we need modified T7 promoters with even higher efficiency of protein expression to meet specific demands. Hence, we tried to transform the wild-type T7 promoter to get modified T7 promoters with increased strength .


T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+ 1) and extends from -17 to +6. As reported in some papers, the sequence specificty of T7 promoter is so strong that some point mutations between positions -11 and -7 may make T7 promoter fail to work. Thus, with the help of previous research, we carefully chose the site which would be mutated by PCR. These sites mainly distribute in the range from -4 to +6. The sequences of these modified promoters are shown in the Table below.


Sequences of Modified Promoters
Part Number Sequence(-17~+6)
BBa_R0085(wild type) TAATACGACTCACTATAGGGAGA
BBa_K2721000 TAATACGACTCACTATCGCGGAG
BBa_K2721001 TAATACGACTCACTCCAGCAATC
BBa_K2721002 TAATACGACTCACTTCAGCGACC
BBa_K2721003 TAATACGACTCACACGAGCGAGA
Test

To test the function of mutant promoters, we chose the GFP as our reporter. By assessing the absolute fluorescence units(RFU) and OD600, we can conclude the relative strength of all promoters. When the E.coli BL21(DE3) is cultured at the stage of logarithmic phase, we added IPTG to induce the expression of GFP in strains BL21(DE3) for 4 hours. And the result is shown as Figure and Table below.

Fig.1 Relative Strength of wildtype T7 promoter and mutant promoters
Part Number Relative Strength
BBa_R0085(wild type) 1
BBa_K2721000 20.99
BBa_K2721001 17.75
BBa_K2721002 7.63
BBa_K2721003 13.92


As we can see from the figure, our mutant promoters showed largely increased strength compared with wild type T7 promoter. Therefore, our mutant promoters offer users more opportunity to control the expression of protein using T7 promoter and permit higher levels of target protein expression to be obtained.

References

[1] Ikeda R A, Ligman C M, Warshamana S, et al. T7 promoter contacts essential for promoter activity in vivo[J]. Nucleic Acids Research, 1992, 20(10): 2517-2524.

[2] Paul S, Stang A, Lennartz K, Tenbusch M, Uberla K. Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution[J]. Nucleic Acids Research, 2013, 41(1):e29.