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

Latest revision as of 03:29, 22 October 2019

description

Overview

UESTC-China knows how important safety is to the entire project, so we divide Safety into two parts: biosafety and Lab Safety. In the biosafety section, we designed a double sterilization system consisting of a light-controlled suicide system and UV sterilization technology to ensure biosafety. Meanwhile we understand the inherent risks of working in a lab facility and aims to take all necessary precautions to ensure no personal or environmental harm occurs. To this end, we have implemented the Lab Safety.

Biosafety

Introduction

In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineered killing measure - double safety sterilization system. In the first security mechanism, we adopted a photo-activated suicide switch based on the YF1-Fix blue-sensitive system to ensure that E.coli can only survive under blue light conditions and be killed in the dark [1]; in the second security mechanism, we used the UV sterilization technology to kill all harmful bacteria and ensure that no other super bacteria are produced. With such a security system, biosafety can be guaranteed to the greatest extent. Further, to avoid, prevent and minimize the information coded by the antibiotic resistance being passed onto the environment that known as horizontal gene transfer, we can use semantic containment methods to achieve a more safe and effective biological containment.

Parts

This is our first security mechanism: BBa_K3034012. It consists of the blue-sensitive promoter switch (BBa_K592004, BBa_K592005 and BBa_K2277233) and a lysin gene named Lysep3-D8(BBa_K3034004), a compound protein which can lyse bacteria from the inside and outside of cells [2], and theoretically has a stronger effect of lysing bacteria.

Experiments

First, amplifying Lysep3-D8 gene from a plasmid containing BBa_K3034012, we inserted it into an expression vector induced by IPTG(final concentration is 0.5mM) and transformed the recombinant plasmid into

E.coli

BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of

E.coli

to verify the effect of cleavage of Lysep3-D8 (Fig. 1).

Second, we inserted red fluorescent protein (TagRFP) after the light-sensitive promoter system to characterize the function of the light control system. The control group was under continuous illumination at 470 nm and the experimental group was in absolute dark conditions(all other conditions are exactly the same). After 24 h, comparing the experimental group with the control group, we found that the experimental group did not show the expected red color visible to the naked eye. In the future, we are going to optimize the light-controlled promoter with methods such as site-specific mutation.

Results

Result of the first experiment:

Fig. 1. Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C) in E.coli BL21(DE3). The experimental data comes from two sets of biological replicates, repeated three times in each group.

Our experimental data shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in OD600 between the experimental group (IPTG+) and the control group (IPTG-) was obvious (Fig. 1). That is, the Lysep3-D8 protein obviously inhibited the growth of

E.coli

(BL21) (inhibition rate was about 30.7%).

Finally, combined with the second safety mechanism-UV sterilization mechanism, we have minimized the risk of leakage of engineered bacteria and production of harmful bacteria. We provide meaningful advice for hardware in biosafety design.

Horizontal Gene Transfer(HGT)

To minimize the risks associated with bacterial release, we'd better promise the biosafety from three mechanisms, mutagenic drift, environmental supplementation and horizontal gene transfer (HGT). At present, there are three main solutions to the problem of horizontal gene transfer of genetically modified organisms: nutritional containment, heteronuclear acid and semantic containment [3]. The control of nutrient containment may be attenuated or invalidated by the supplementation of natural compounds. The heterologous nucleic acid method is difficult to artificially insert artificially synthesized bases in biological nucleic acids, so we will use semantic containment methods to further control our engineered

E.coli

in the future.

Its main principle is to transform the genetic sequence of the organism so that the UAG triplet codon is no longer a stop codon, but can encode an amino acid. In order to ensure a high probability of low leakage. UAG is encoded into artificially modified amino acids, rather than natural amino acids, are effective [4]. Studies have shown that this method is achievable and very powerful, so we believe that the same method can be used to prevent the leakage of resistance genes in our experiments, and the essential genes of

E.coli

such as the enzyme III subunit δ (holB), methionyl-tRNA synthetase (metG), phosphoglycerate kinase (pgk), etc. can be modified to encode the codon UAG into NSAA L-4, 4'-biphenylalanine (bipA), which has a different size and geometry than any standard amino acid, as well as hydrophobic chemicals that are expected to be compatible with the protein core.

References

[1]Wang, G., Lu, X., Zhu, Y., et al. (2018). A light-controlled cell lysis system in bacteria. Journal of industrial microbiology & biotechnology, 45(6), 429-432.
[2]Wang, S., Gu, J., Lv, M. et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. Journal of Microbiology, 55: 403.
[3]Marliere P. (2009). The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world. Systems and Synthetic Biology, 3(1-4):77–84.
[4]Daniel J. Mandell, Marc J. Lajoie, Michael T. Mee, et al. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518: 55-60.

Lab Safety

Chasiss

The chassis we used are

E.coli

DH5a,

E.coli

BL21(DE3) ,

E.coli

CICIM B0016,

E.coli

TOP 10 and

E.coli

MC1061, which all belong to RISK GROUP 1, means they are low risk for human being and environment.

Part

Our parts are all taken from the RISK GROUP 1 and RISK GROUP 2 organisms. The RISK GROUP 1 organisms are not safe to cause diseases in healthy adults. Although the RISK GROUP 2 organisms may cause diseases in humans, the parts we extracted were all synthesized from the company, without any risk of pathogens.

Expected Protection Mechanism

The greater risk of our project is that genetically modified bacteria that carry antibiotic resistance can escape into the wild under incorrect operation. Although the transgenic bacteria we used don't cause disease themselves, they can still transfer plasmids that carry antibiotic resistance to those pathogens. Therefore, UV disinfection lamps are installed in our hardware devices for biosafety. (see more at Biosafety and Hardware)

Disposal

To avoid the pollution of environment, we gather all cultivate medium with bacteria and sterilize them before discarding. For some reagents with health risk, such as TEMED, we use them carefully with the correct position and keep another separate container for hazardous waste. In order to keep our laboratory safe and clean, we carry out regular cleaning, sterilization, and disinfection every day. Besides, we also check out the electrical installment and instrument to ensure safe experiment and environment. Ultra-clean working table is treated with ultraviolet sterilization before use.

Training

We had a lesson about lab safety provided by the university biosafety office in our freshman year. In this lesson, freshman can learn the correct experimental operation and much knowledge about chemical drugs and reagents. And the students who pass the final safety test will accept a diploma, with which one may enter the lab and start experiment.

Operation

Our experimental operation strictly in accordance with the guidelines:

Laboratory coveralls, gowns or uniforms must be worn at all times for work in the laboratory;
When some volatile toxic reagents are necessary, we will operate in the fume hood; All reagents have designated position and must returned after experiment;
A variety of drugs and reagents must be signed a clean label including the name, concentration, specification, etc.;
Daily decontamination of all work surfaces when work is complete;
Prohibition of food, drink and smoking materials in lab setting;
Pipetting by mouth of any material is forbidden. You must always use the teats, syringes, and pipette-fillers provided;
Contaminated glassware, plastic ware, microscope slides and discarded Petri dishes etc., must be placed in the receptacles indicated by the lecturer in charge;

IGEM Safety Form

Final Safety Form

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 <div class="firstless" >
    	<ul class="f_mh mya"  style="display: none;margin-left:5px">
-                 <li><a>Overview</a></li>
+                 <li><a href="#title_1">Overview</a></li>
-		<li><a class="ajs" >Biosafety<b class="caret"></b></a>
+		<li><a class="ajs" href="#title_2">Biosafety<b class="caret"></b></a></li>
 			<ul class="secondli">
 				<li style="padding-top:10px;"><a href="#stitle_1">Introduction</a></li>
@@ Line 68: / Line 53: @@
 				<li><a href="#stitle_6">References</a></li>
 			</ul>
-		</li>
-		<li><a class="ajs">Lab Safety<b class="caret"></b></a></li>
+		<li><a class="ajs" href="#title_3">Lab Safety<b class="caret"></b></a></li>
 			<ul class="secondli">
 				<li style="padding-top:10px;"><a href="#stitle_7">Chasiss</a></li>
@@ Line 85: / Line 69: @@
 <div class="container col-7" id="divHp">
    <div class="col-fmh" >
+<div class="part">
-     <div class="bigtitle">
+     <div class="bigtitle xlj" id="title_1">
            <p>Overview</p>
      </div>
-     <div class="mainbody" style="text-indent:50px;">
+     <div class="mainbody">
-          UESTC-China knows how important safety is to the entire project, so we divide Safety into two parts: biosafety and Lab Safety. In the biosafety section, we designed a light-controlled suicide switch and finally used a more economical and efficient UV sterilization on the hardware to ensure safety. Meanwhile we understand the inherent risks of working in a lab facility and aims to take all necessary precautions to ensure no personal or environmental harm occurs. To this end, we have implemented the Lab Safety.
+          UESTC-China knows how important safety is to the entire project, so we divide Safety into two parts: biosafety and Lab Safety. In the biosafety section, we designed a double sterilization system consisting of a light-controlled suicide system and UV sterilization technology to ensure biosafety. Meanwhile we understand the inherent risks of working in a lab facility and aims to take all necessary precautions to ensure no personal or environmental harm occurs. To this end, we have implemented the Lab Safety.
      </div>
+</div>
 <div class="part">
-		<div class="bigtitle">
+		<div class="bigtitle xxlj" id="title_2">
 			<p>Biosafety</p>
 		</div>
@@ Line 101: / Line 85: @@
 		   <h2 id="stitle_1">Introduction</h2>
-		<div class="mainbody" style="text-indent: 50px;">
+		<div class="mainbody">
-			In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineering kill measure——light-activated suicide switches based on the YF1-Fix blue-sensitive system. It ensures that engineering bacteria can survive only in devices where blue light is present. However, our experiments showed that  the activation of the light control system requires a long dark time, which is contrary to our concept of efficient ciprofloxacin degradation, so we decided to adopt a more efficient, faster and less costly UV sterilization system. In fact, our hardware systems are also easier to achieve UV sterilization than light-controlled cracking. Besides, to avoid, prevent and minimize the information coded by the antibiotic resistance being passed onto the environment that known as horizontal gene transfer, we can use semantic containment methods to achieve a more safe and effective biological containment.
+			In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineered killing measure - double safety sterilization system. In the first security mechanism, we adopted a photo-activated suicide switch based on the YF1-Fix blue-sensitive system to ensure that <i>E.coli</i> can only survive under blue light conditions and be killed in the dark [1]; in the second security mechanism, we used the UV sterilization technology to kill all harmful bacteria and ensure that no other super bacteria are produced. With such a security system, biosafety can be guaranteed to the greatest extent. Further, to avoid, prevent and minimize the information coded by the antibiotic resistance being passed onto the environment that known as horizontal gene transfer, we can use semantic containment methods to achieve a more safe and effective biological containment.
 		</div>
 			<h2 id="stitle_2">Parts</h2>
-		<div class="mainbody" style="text-indent: 50px;">
+		<div class="mainbody">
-			This is our initial design: <a href="http://parts.igem.org/Part:BBa_K3034012">BBa_K3034012</a>. It consists of the blue-sensitive promoter switch (<a href="http://parts.igem.org/Part:BBa_K592004">BBa_K592004</a>, <a href="http://parts.igem.org/Part:BBa_K592005">BBa_K592005</a> and <a href="http://parts.igem.org/Part:BBa_K2277233">BBa_K2277233</a>) and a lysin gene named Lysep3-D8(<a href="http://parts.igem.org/Part:BBa_K3034004">BBa_K3034004</a>), a compound protein which can lyse bacteria from the inside and outside of cells[1], and theoretically has a stronger effect of lysing bacteria. In the latest design, we used an ultraviolet sterilization device instead of a suicide system.
+			This is our  first security mechanism: <a href="http://parts.igem.org/Part:BBa_K3034012">BBa_K3034012</a>. It consists of the blue-sensitive promoter switch (<a href="http://parts.igem.org/Part:BBa_K592004">BBa_K592004</a>, <a href="http://parts.igem.org/Part:BBa_K592005">BBa_K592005</a> and <a href="http://parts.igem.org/Part:BBa_K2277233">BBa_K2277233</a>) and a lysin gene named Lysep3-D8(<a href="http://parts.igem.org/Part:BBa_K3034004">BBa_K3034004</a>), a compound protein which can lyse bacteria from the inside and outside of cells [2], and theoretically has a stronger effect of lysing bacteria.
 		</div>
 			<h2 id="stitle_3">Experiments</h2>
 		<div class="mainbody">
-			Based on these characteristics, we designed the following experiment:
 		</div>
-		<div class="mainbody" style="text-indent: 50px;">
+                <br>
-			First, amplifying Lysep3-D8 gene from a plasmid containing <a href="http://parts.igem.org/Part:BBa_K3034012">BBa_K3034012</a>, we inserted it into an expression vector induced by IPTG(final concentration is 0.5mM) and transformed it into <p style="display: inline;font-style: italic">E.coli</p> BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of <p style="display: inline;font-style: italic">E.coli</p> to verify the effect of cleavage of Lysep3-D8(Fig.1).
+		<div class="mainbody">
-<p>Second, we inserted red fluorescent protein (TagRFP) after the light-sensitive promoter system to characterize the function of the light control system. The control group was under continuous illumination at 470 nm and the experimental group was in absolute dark conditions(all other conditions are exactly the same.). After 24 h, comparing the experimental group with the control group, we found that the experimental group did not show the expected red color visible to the naked eye.</p>
+			First, amplifying Lysep3-D8 gene from a plasmid containing <a href="http://parts.igem.org/Part:BBa_K3034012">BBa_K3034012</a>, we inserted it into an expression vector induced by IPTG(final concentration is 0.5mM) and transformed the recombinant plasmid into <p style="display: inline;font-style: italic">E.coli</p> BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of <p style="display: inline;font-style: italic">E.coli</p> to verify the effect of cleavage of Lysep3-D8 (Fig. 1). <br>
-<p>Finally, we carried out the bactericidal effect of ultraviolet rays irradiated at different gradient times.In the experiment to verify the ultraviolet bactericidal effect, we designed the experimental group with the duration of UV irradiation (10 mins, 30 mins, 60 mins), and did not irradiate ultraviolet light as a blank control group. At different irradiation times, 20 microliters of bacterial solution was taken and applied to LB medium, and cultured overnight, which can be used to judge the ultraviolet sterilization effect (Fig.2).</p>
+<p>Second, we inserted red fluorescent protein (TagRFP) after the light-sensitive promoter system to characterize the function of the light control system. The control group was under continuous illumination at 470 nm and the experimental group was in absolute dark conditions(all other conditions are exactly the same). After 24 h, comparing the experimental group with the control group, we found that the experimental group did not show the expected red color visible to the naked eye. In the future, we are going to optimize the light-controlled promoter with methods such as site-specific mutation.</p><br>
 		</div>
@@ Line 127: / Line 110: @@
 		  <div class="picture">
-			  <img src="https://static.igem.org/mediawiki/2019/8/81/T--UESTC-China--safety1.png" alt="logo" width="60%">
+			  <img src="https://static.igem.org/mediawiki/2019/8/81/T--UESTC-China--safety1.png" alt="logo" width="100%">
 		  </div>
-		<div class="words">Fig.1. Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C). The experimental data comes from two sets of biological replicates, repeated three times in each group. </div>
+		<div class="words">Fig. 1. Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C) in <i>E.coli</i> BL21(DE3). The experimental data comes from two sets of biological replicates, repeated three times in each group. </div>
-		<div class="mainbody" style="text-indent: 50px">
-			Figure 1 shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in Abs600 between the experimental group (+IPTG) and the control group (-IPTG) was larger and larger. That is, the Lysep3-D8 protein inhibited the growth of <p style="display: inline;font-style: italic">E.coli</p> (BL21) (inhibition rate was about 30.7%).
-		  </div>
 		<div class="mainbody">
-			Result of the last experiment：
+			Our  experimental data shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in OD600 between the experimental group (IPTG+) and the control group (IPTG-) was obvious (Fig. 1). That is, the Lysep3-D8 protein obviously inhibited the growth of <p style="display: inline;font-style: italic">E.coli</p> (BL21) (inhibition rate was about 30.7%).
-		</div>
+		  </div><br>
+		<div class="mainbody">
-		  <div class="picture">
-			  <img src="https://static.igem.org/mediawiki/2019/2/22/T--UESTC-China--DH5α_10min.png" alt="logo" width="60%">
-		  </div>
-	        <div class="words">Fig.2.1. Characterization of the sterilization effect of the ultraviolet sterilization lamp after irradiated for 10 minutes. The left picture shows the control group (no UV irradiation at room temperature), and the right picture shows the experimental group (UV lamp with a power of 8 watts at room temperature for 10 minutes). </div>
-<div class="picture">
-			  <img src="https://static.igem.org/mediawiki/2019/2/22/T--UESTC-China--DH5α_30min.png" alt="logo" width="60%">
-		  </div>
-	        <div class="words">Fig.2.2. Characterization of the sterilization effect of the ultraviolet sterilization lamp after irradiated for 30 minutes. The left picture shows the control group (no UV irradiation at room temperature), and the right picture shows the experimental group (UV lamp with a power of 8 watts at room temperature for 30 minutes). </div>
-	      <div class="mainbody">
-			  Result of the last experiment：
-	      </div>
-		  <div class="picture">
-			  <img src="https://2019.igem.org/File:T--UESTC-China--DH5α_10min.png" alt="logo" width="60%">
-		  </div>
-		<div class="words">
+<p>Finally, combined with the second safety mechanism-UV sterilization mechanism, we have minimized the risk of leakage of engineered bacteria and production of harmful bacteria. We provide meaningful advice for hardware in biosafety design.</p>
-		On the left side of the figure, the bacterial cultures of the blank control were cultured for 10 mins, 30 mins, and 60 min, and the plate effect diagram was applied. The right side of the figure was the sterilization effect of the bacterial solution coating plate after the ultraviolet irradiation at the same time interval.</div>
-		<div class="mainbody" style="text-indent: 50px;">
-<p>From the above experimental results, under the condition of suitable protein expression, the antibacterial effect of Lysep3-D8 protein induced by IPTG strong inducer was not significant compared with the blank control. When we verified the induction intensity of the blue light-controlled promoter, from the experimental results, it did not have a good induction effect compared to the IPTG-inducible promoter. And from the above experiments using different durations of UV irradiation, we can conclude that UV has a significant bactericidal effect and can be easily applied to our hardware equipment.</p>
-<p>In order to realize our concept of high efficiency and biosafety, in the realization of hardware biosafety, we finally decided to adopt a low-cost and high-efficiency physical sterilization method, using double-ultraviolet sterilization to prevent engineering bacteria leakage to the maximum extent to ensure the environment. And people's safety. This can promote the further development of hardware, making it more potential to enter the community.</p>
 		</div>
@@ Line 170: / Line 126: @@
 		 <h2 id="stitle_5">Horizontal Gene Transfer(HGT)</h2>
-		<div class="mainbody" style="text-indent: 50px">
+		<div class="mainbody">
-			To minimize the risks associated with bacterial release, we'd better promise the biosafety from three mechanisms mutagenic drift, environmental supplementation and horizontal gene transfer (HGT). At present, there are three main solutions to the problem of horizontal gene transfer of genetically modified organisms: nutritional containment, heteronuclear acid and semantic containment[2]. The control of nutrient containment may be attenuated or invalidated by the supplementation of natural compounds. The heterologous nucleic acid method is difficult to artificially insert artificially synthesized bases in biological nucleic acids, so we will use semantic containment methods to futher control our engineered <p style="display: inline;font-style: italic">E.coli</p> in the future. <br>
+			To minimize the risks associated with bacterial release, we'd better promise the biosafety from three mechanisms, mutagenic drift, environmental supplementation and horizontal gene transfer (HGT). At present, there are three main solutions to the problem of horizontal gene transfer of genetically modified organisms: nutritional containment, heteronuclear acid and semantic containment [3]. The control of nutrient containment may be attenuated or invalidated by the supplementation of natural compounds. The heterologous nucleic acid method is difficult to artificially insert artificially synthesized bases in biological nucleic acids, so we will use semantic containment methods to further control our engineered <p style="display: inline;font-style: italic">E.coli</p> in the future. <br>
-		</div>
+		</div>	<br>
-			<div  class="mainbody" style="text-indent: 50px">
+			<div  class="mainbody">
-				Its main principle is to transform the genetic sequence of the organism so that the UAG triplet codon is no longer a stop codon, but can encode an amino acid. In order to ensure a high probability of low leakage. UAG is encoded into artificially modified amino acids, rather than natural amino acids, are effective[3]. Studies have shown that this method is achievable and very powerful, so we believe that the same method can be used to prevent the leakage of resistance genes in our experiments, and the essential genes of <p style="display: inline;font-style: oblique">E.coli</p> such as the enzyme III subunit δ (holB), methionyl-tRNA synthetase (metG), phosphoglycerate kinase (pgk), etc. can be modified to encode the codon UAG into NSAA L-4, 4'-biphenylalanine (bipA), which has a different size and geometry than any standard amino acid, as well as hydrophobic chemicals that are expected to be compatible with the protein core.
+				Its main principle is to transform the genetic sequence of the organism so that the UAG triplet codon is no longer a stop codon, but can encode an amino acid. In order to ensure a high probability of low leakage. UAG is encoded into artificially modified amino acids, rather than natural amino acids, are effective [4]. Studies have shown that this method is achievable and very powerful, so we believe that the same method can be used to prevent the leakage of resistance genes in our experiments, and the essential genes of <p style="display: inline;font-style: oblique">E.coli</p> such as the enzyme III subunit δ (holB), methionyl-tRNA synthetase (metG), phosphoglycerate kinase (pgk), etc. can be modified to encode the codon UAG into NSAA L-4, 4'-biphenylalanine (bipA), which has a different size and geometry than any standard amino acid, as well as hydrophobic chemicals that are expected to be compatible with the protein core.
 			</div>
@@ Line 182: / Line 138: @@
 	   <h2 id="stitle_6">References</h2>
-		  <div class="mainbody">[1] Shuang Wang, Jingmin Gu, Meng Lv, et al. <p style="display: inline;font-style: italic"> The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region.</p> Journal of Microbiology, 2017, Volume 55, Number 5, Page 403<br>
+		  <div class="mainbody">[1]Wang, G., Lu, X., Zhu, Y., et al. (2018). A light-controlled cell lysis system in bacteria. <i>Journal of industrial microbiology & biotechnology, 45</i>(6), 429-432.<br>
-[2] Philippe Marliere. <p style="display: inline;font-style: italic"> The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world.</p> Systems and Synthetic Biology, 2009, Volume 3, Number 1-4, Page 77<br>
+[2]Wang, S., Gu, J., Lv, M. et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. <i>Journal of Microbiology</i>, 55: 403.<br>
-[3] Daniel J. Mandell, Marc J. Lajoie, Michael T. Mee, et al. <p style="display: inline;font-style: italic"> Biocontainment of genetically modified organisms by synthetic protein design.</p> Nature, 2015, Volume 518, Pages 55–60
+[3]Marliere P. (2009). The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world. <i>Systems and Synthetic Biology, 3</i>(1-4):77–84.<br>
+[4]Daniel J. Mandell, Marc J. Lajoie, Michael T. Mee, et al. (2015). Biocontainment of genetically modified organisms by synthetic protein design. <i>Nature, 518</i>: 55-60.
 </div>
 	 </div>
 	<div class="part">
-		<div class="bigtitle">
+		<div class="bigtitle xxlj" id="title_3">
 			<p>Lab Safety</p>
 		</div>
@@ Line 195: / Line 152: @@
 		<h2 id="stitle_7">Chasiss</h2>
-		<div class="mainbody" style="text-indent: 50px;">
+		<div class="mainbody">
 			The chassis we used are<p style="display: inline;font-style: italic"> E.coli </p>DH5a, <p style="display: inline;font-style: italic"> E.coli </p> BL21(DE3) , <p style="display: inline;font-style: italic"> E.coli </p>CICIM B0016, <p style="display: inline;font-style: italic"> E.coli </p>TOP 10 and <p style="display: inline;font-style: italic"> E.coli </p>MC1061, which all belong to RISK GROUP 1, means they are low risk for human being and environment.
 		</div>
@@ Line 208: / Line 165: @@
 		<div class="mainbody">
-			The greater risk of our project is that genetically modified bacteria that carry antibiotic resistance can escape into the wild under incorrect operation. Although the transgenic bacteria we used don't cause disease themselves, they can still transfer plasmids that carry antibiotic resistance to those pathogens. Therefore, UV disinfection lamps are installed in our hardware devices for biosafety.(see more at <a href="#title_1">Biosafety</a> and <a href="https://2019.igem.org/Team:UESTC-China/Hardware">Hardware</a>)
+			The greater risk of our project is that genetically modified bacteria that carry antibiotic resistance can escape into the wild under incorrect operation. Although the transgenic bacteria we used don't cause disease themselves, they can still transfer plasmids that carry antibiotic resistance to those pathogens. Therefore, UV disinfection lamps are installed in our hardware devices for biosafety. (see more at <a href="#title_1">Biosafety</a> and <a href="https://2019.igem.org/Team:UESTC-China/Hardware">Hardware</a>)
 		</div>
 		<h2 id="stitle_10">Disposal</h2>
-		<div class="mainbody" style="text-indent: 50px;">
+		<div class="mainbody">
 		To avoid the pollution of environment, we gather all cultivate medium with bacteria and sterilize them before discarding. For some reagents with health risk, such as TEMED, we use them carefully with the correct position and keep another separate container for hazardous waste. In order to keep our laboratory safe and clean, we carry out regular cleaning, sterilization, and disinfection every day. Besides, we also check out the electrical installment and instrument to ensure safe experiment and environment. Ultra-clean working table is treated with ultraviolet sterilization before use.
 		</div>
@@ Line 223: / Line 180: @@
 		<h2 id="stitle_11">Training</h2>
-		<div class="mainbody" style="text-indent: 50px;">
+		<div class="mainbody">
 		We had a lesson about lab safety provided by the university biosafety office in our freshman year. In this lesson, freshman can learn the correct experimental operation and much knowledge about chemical drugs and reagents. And the students who pass the final safety test will accept a diploma, with which one may enter the lab and start experiment.
 		</div>
@@ Line 238: / Line 195: @@
 		<div class="mainbody">
-			Our experimental operation strictly in accordance with the guidelines:<br>
+			Our experimental operation strictly in accordance with the guidelines:<br><br>
 Laboratory coveralls, gowns or uniforms must be worn at all times for work in the laboratory;<br>
 When some volatile toxic reagents are necessary, we will operate in the fume hood;
@@ Line 263: / Line 220: @@
 		var oli2=$(".secondli li");
 		var oS=$(".secondli");
+		var oli=$(".f_mh>li");
 		$(window).scroll(function(){
-			$(".bigtitle:first").each(function(){//到第一个大标题折叠后面的
+			$(".xlj").each(function(){//无分级大标题隐藏其他次级标题
 				var _target=parseInt($(this).offset().top-$(window).scrollTop()-_line);
 				if (_target<=0) {
@@ Line 271: / Line 229: @@
 				}
 			});
-			$(".part").each(function(index){
+			$(".xxlj").each(function(index){//有分级对应滚动展开
 				var _target=parseInt($(this).offset().top-$(window).scrollTop()-_line);
 				var _i=index;
@@ Line 282: / Line 240: @@
 			});
-			$("h2").each(function(index){
+			$(".bigtitle").each(function(index){//对应大标题高亮
+				var _target=parseInt($(this).offset().top-$(window).scrollTop()-_line);
+				var _i=index;
+				if (_target<=0) {
+					oli.removeClass('active');
+					oli.eq(_i).addClass('active');
+				}
+			});
+			$("h2").each(function(index){//对应次级标题高亮
 				var _target=parseInt($(this).offset().top-$(window).scrollTop()-_line);
 				var _i=index;
@@ Line 297: / Line 264: @@
 		});
-           $('.f_mh li a').click(function() {
+           $('.f_mh li a').click(function() {//点击缓动
              $('html, body').stop().animate({
                  scrollTop: $($(this).attr('href')).offset().top - 100}, 1000);
              return false;
          });
-           $('.mainbody a').click(function() {
+           $('.mainbody a').click(function() {//点击缓动
              $('html, body').stop().animate({
                  scrollTop: $($(this).attr('href')).offset().top - 100}, 1000);
@@ Line 313: / Line 280: @@
 	<script>
-		var oA=document.getElementsByClassName("ajs");
+		var oA=document.getElementsByClassName("ajs");//点击侧边栏大标题对应展开次级标题
 		var oS=document.getElementsByClassName("secondli")
          for(var i=0;i<oA.length;i++)
@@ Line 330: / Line 297: @@
 <script>
-$(document).ready(function() {
+$(document).ready(function() { //侧边栏隐藏
          $(window).scroll(function() {