<p>In this part, we designed a bi-directional transcription system which is regulated by light. It can express toxicity or attractant in different conditions. Specifically, we linked poison protein genes and attractant gene to different sites of reversible promoter(P<sub>con</sub>). This reversible promoter can change the direction with the action of int protein, whose expression is regulated by PompR, a promoter regulated by light. To be specific, an enzyme encoded by ho1 gene can catalyze the synthesis of BV protein, which can catalyze another protein which is encoded by pcya gene to synthesize PCB protein. PCB protein can combine with cph8 protein and form cph8-PCB complex. This complex is easy to be phosphorylated under normal conditions. The result of phosphorylation is ompR protein can segregate from the complex and combine with P<sub>ompr</sub> and promote the expression of int protein. The specific numbers of biobricks are listed at the Design part of the Project.</p>
+
<p>To guarantee biosafety, our team takes three measures as follow:
−
<p>The mechanism of this system is that in the condition of light, cph8-PCB complex cannot be phosphorylated, correspondingly, ompR protein cannot combine with P<sub>ompR</sub>, so int protein is fail to be synthesized.</p>
+
</p>
−
<p>So, our system’s mechanism of action is that we use light with certain wavelength to irradiate the system in initial, in order to inhibit the expression of int protein and on the contrary, express the attractant. When the attractant accumulate to a certain concentration, we will move away the source of light, in order to express int protein, so that the system can transcribe in the opposite direction to express poison protein. The specific mechanism of this system is shown in the figure below. (<a href="#figure-1">Figure 1</a>)</p>
+
<p>Firstly, we designed a device to cultivate <em>E.coli</em> inside and restrict their movement at the same time, thus preventing the difussion of our engineering bacteria to soil.
−
<figure id="figure-1">
+
</p>
−
<img src="img/team/figure-1.png" alt="figure1" />
+
<p> In addition, we established a photoinduced bidirectional transcription system regulated by light, where we can divide the process of attracting and killing nematodes into two periods to realize the relatively timing and quantitative release of the toxic proteins, bringing down the retention in the environment.
−
<figcaption>Figure 1</figcaption>
+
</p>
−
</figure>
+
<p>Thirdly, a suicide system was constructed based on the principle of quorum sensing in microorganisms which would enable us to control the bacterial colony density artificially and therefore the quantity of engineering bacteria can be maintained in a stable and controllable range.
−
<p>In this system, we can regulate the expression degree through the bi-directional transcription mechanism which is regulated by light. The meaning of this system is that we can avoid the environmental or biological hazards caused by the high expression of poison protein and attractant at the same time.
+
</p>
−
</p>
+
<P>Below indicated the specific principles and designs.
−
+
</P>
−
<h2>Suicide</h2>
+
−
<p>We not only considered the potential safety hazard caused by the high expression of poison protein and attractant, but also designed a suicide system for our engineering bacteria, in order to prevent its spread in the environment and mass propagation.
+
−
</p>
+
−
<p>The mechanism of our suicide switch is that engineering bacteria is sensitive to the population size. Our system contains a section of luxI gene, and a synthase encoded by it is able to catalyze the synthesis of AHL molecule. AHL is a kind of organic small molecule which is able to across the membrane freely. Another gene contained in our system called LuxR, it encodes LuxR protein, which can combine with AHL molecule. When AHL molecule reaches a certain concentration, LuxR protein will combine with PluxR promoter, and activate the expression of MazF poison protein, thus make our bacteria suicide themselves. The specific numbers of biobricks are listed at the Module3 part of the Project.
+
−
</p>
+
−
<p>We can see that the quantity of bacteria can maintain in a relatively stable condition through this system, in order to limit the propagation of our engineering bacteria which have antibiotic resistance.
+
−
</p>
+
−
+
−
<h2>Lab&Environment safety</h2>
+
−
<p>We have a strict safety regulations in our laboratory. The rules are as follows. When we enter the laboratory, all the members are required to wear the lab-gown and the latex examination gloves. During our experiment, such as the bacterination, we have to do it in the Clean Bench. And when we have to use the hazardous reagents, we must do it in the fuming cupboard as the safety regulations told. Moreover, the reagents we used should be poured into disposal bottle. And the mediums must be throw after doing the ultra-high-temperature sterilizing. In addition, the trash must be classified to throw in the normal trash can or the medical waste can.
+
−
</p>
+
−
<p>As for the environment safety, we also considerate how our engineering bacteria works. We plan to design a semienclosed device to cultivate engineering bacteria to trap and kill the nematodes by modeling. So it avoids the security risk of direct contact of the soil and the bacteria. Furthermore, as we said before, we designed the suicide system to avoid the excess propagation of the recombinant bacteria.
+
−
</p>
+
−
<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>
+
<h2>Device
+
</h2>
+
<p>Considering the biosafety, we improved device 2.0 into a relatively closed unit, and we also designed a dome to cover the medium. In addition, replacement and remove of medium and engineering bacteria would all be done in the labs or safety rooms of factories which guarantees relative closure of the device used in farmland and avoids engineering bacteria spreading
+
</p>
−
<h4>Safe Shipment</h4>
+
<h2>Switch</h2>
+
<p>Although Bace16 and rMpL are both toxin proteins towards nematodes specially, the over expression of these proteins can also break the ecological equilibrium inevitably. Therefore we build a photoinduced bidirectional transcription system to make the expression of toxin proteins under control so that our engineered bacteria can express attractants or toxin proteins in different conditions. This system can be divided into three main parts: photoinduced system, reversible transcription system and bait-killer system.
+
</p>
−
<p>Did you face any safety problems in sending your DNA parts to the Registry? How did you solve those problems?</p>
+
<h3>1. Photoinduced system
+
</h3>
+
<p>The red light sensor (Cph8) is a fusion protein which consists of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is a member of the plant photoreceptor family. With the biosynthesis of PCB, Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Under the exposure of red light, however, Cph1 is deactivated, inhibiting the autophosphorylation, thus turning off gene expression. In order to regulate the direction of promoter J23110 under the light signal, gp35, an integrase is added upstream the PompC promoter.</p>
+
<h3>2. Directional reverse system
+
</h3>
+
<p>We construct two main circuits. The first one expresses gp35 serine integrase, which can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP, changing the two sites to attL and attR at the meantime. This inversion can be reversible by appropriately controlling the conditional expression of integrase and an excisionase in Bxb1 named gp47 at certain ratio. </p>
<p>The second one contains a switch promoter(J23110), two functional genes(there GFP and RFP), two RBS and terminators. At first, the plasmid expresses GFP. When gp35 is expressed, the switch will turn around and RFP on the other side of the plasmid is going to be expressed.
<p>We replaced GFP and RFP into limonene synthase and toxic protein respectively, so the release of the two proteins can be regulated by gp35 directly, and regulated by the light signals indirectly.
+
</p>
+
<p><a href="https://2015.igem.org/Team:BNU-CHINA/Circuit_Design">For more information, please see our circuit design section.</a>
+
</p>
+
<p>Generally speaking, the Switch system works in this way--we keep treating the system with light of wavelength in 600nm at first so gene gp35 will be repressed and the system will express the chemical attractants like limonene. While when the concentration of attractants reaches a certain point (see modeling part), we turn off the light to trigger the expressing of protein gp35, so the system will work in a reverse direction and the toxin protein will be expressed. We can conclude that the significance of our system is we build a controllable bidirectional light regulated system to avoid expressing baits and toxin proteins simultaneously and strongly so that we can avoid the harm that the system may do to the environment.
+
</p>
+
<h2>Suicide
+
</h2>
+
<p>We not only considered the potential safety problem caused by the high expression of the toxic protein and attractant, but also designed a suicide system for our engineering bacteria to solve the problem by regulating the population size of our bacteria.
+
</p>
+
<p>We build our system based on the phenomenon of quorum sensing. Synthase LuxI coded by gene luxI can catalyze the synthesis of AHL, which is a kind of organic small molecule able to across the membrane freely. Also, protein LuxR encoded by gene luxR can bind with AHL molecules to form a complex. This complex can bind with promoter luxpR, after which the transcription of mazf gene in the downstream will be triggered and bacteria will be killed by toxin protein MazF. The related parts have been shown in Module 3 of our Project page previously.
+
</p>
+
+
<h2>Lab&Environment safety</h2>
+
<p>To guarantee the lab safety, we follow safety rules strictly. For instance, all team members in the lab should wear clothes for lab-use, wear gloves and work in the super clean bench when necessary; poison reagent should be used in the fuming cupboard; and the waste liquid and medium should be poured or thrown differently.
+
</p>
+
<p> As for environmental safety, on one hand, we design a semi-closed device to avoid the spread of engineering bacteria in which way we could increase the safety level and improve the attracting and killing efficiency of our bacteria; On the other hand, light-regulated bidirectional transcription system can avoid over-expression of toxin proteins, and the design of suicide part can regulate the population density of engineering bacteria. All in all, biosafety is ensured in both the developing and the application stage of our project.
+
+
</p>
</html>
</html>
Revision as of 06:55, 18 September 2015
Team:BNU-CHINA - 2015.igem.org
Safety
Overview
To guarantee biosafety, our team takes three measures as follow:
Firstly, we designed a device to cultivate E.coli inside and restrict their movement at the same time, thus preventing the difussion of our engineering bacteria to soil.
In addition, we established a photoinduced bidirectional transcription system regulated by light, where we can divide the process of attracting and killing nematodes into two periods to realize the relatively timing and quantitative release of the toxic proteins, bringing down the retention in the environment.
Thirdly, a suicide system was constructed based on the principle of quorum sensing in microorganisms which would enable us to control the bacterial colony density artificially and therefore the quantity of engineering bacteria can be maintained in a stable and controllable range.
Below indicated the specific principles and designs.
Device
Considering the biosafety, we improved device 2.0 into a relatively closed unit, and we also designed a dome to cover the medium. In addition, replacement and remove of medium and engineering bacteria would all be done in the labs or safety rooms of factories which guarantees relative closure of the device used in farmland and avoids engineering bacteria spreading
Switch
Although Bace16 and rMpL are both toxin proteins towards nematodes specially, the over expression of these proteins can also break the ecological equilibrium inevitably. Therefore we build a photoinduced bidirectional transcription system to make the expression of toxin proteins under control so that our engineered bacteria can express attractants or toxin proteins in different conditions. This system can be divided into three main parts: photoinduced system, reversible transcription system and bait-killer system.
1. Photoinduced system
The red light sensor (Cph8) is a fusion protein which consists of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is a member of the plant photoreceptor family. With the biosynthesis of PCB, Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Under the exposure of red light, however, Cph1 is deactivated, inhibiting the autophosphorylation, thus turning off gene expression. In order to regulate the direction of promoter J23110 under the light signal, gp35, an integrase is added upstream the PompC promoter.
2. Directional reverse system
We construct two main circuits. The first one expresses gp35 serine integrase, which can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP, changing the two sites to attL and attR at the meantime. This inversion can be reversible by appropriately controlling the conditional expression of integrase and an excisionase in Bxb1 named gp47 at certain ratio.
Fig.1
The second one contains a switch promoter(J23110), two functional genes(there GFP and RFP), two RBS and terminators. At first, the plasmid expresses GFP. When gp35 is expressed, the switch will turn around and RFP on the other side of the plasmid is going to be expressed.
Fig.2
3. Trap and kill system
We replaced GFP and RFP into limonene synthase and toxic protein respectively, so the release of the two proteins can be regulated by gp35 directly, and regulated by the light signals indirectly.
Generally speaking, the Switch system works in this way--we keep treating the system with light of wavelength in 600nm at first so gene gp35 will be repressed and the system will express the chemical attractants like limonene. While when the concentration of attractants reaches a certain point (see modeling part), we turn off the light to trigger the expressing of protein gp35, so the system will work in a reverse direction and the toxin protein will be expressed. We can conclude that the significance of our system is we build a controllable bidirectional light regulated system to avoid expressing baits and toxin proteins simultaneously and strongly so that we can avoid the harm that the system may do to the environment.
Suicide
We not only considered the potential safety problem caused by the high expression of the toxic protein and attractant, but also designed a suicide system for our engineering bacteria to solve the problem by regulating the population size of our bacteria.
We build our system based on the phenomenon of quorum sensing. Synthase LuxI coded by gene luxI can catalyze the synthesis of AHL, which is a kind of organic small molecule able to across the membrane freely. Also, protein LuxR encoded by gene luxR can bind with AHL molecules to form a complex. This complex can bind with promoter luxpR, after which the transcription of mazf gene in the downstream will be triggered and bacteria will be killed by toxin protein MazF. The related parts have been shown in Module 3 of our Project page previously.
Lab&Environment safety
To guarantee the lab safety, we follow safety rules strictly. For instance, all team members in the lab should wear clothes for lab-use, wear gloves and work in the super clean bench when necessary; poison reagent should be used in the fuming cupboard; and the waste liquid and medium should be poured or thrown differently.
As for environmental safety, on one hand, we design a semi-closed device to avoid the spread of engineering bacteria in which way we could increase the safety level and improve the attracting and killing efficiency of our bacteria; On the other hand, light-regulated bidirectional transcription system can avoid over-expression of toxin proteins, and the design of suicide part can regulate the population density of engineering bacteria. All in all, biosafety is ensured in both the developing and the application stage of our project.
