Difference between revisions of "Team:RDFZ-China/Measurement"

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                 <li>
 
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                     <ul>
 
                     <ul>
−
                         <li><a href="#section1">What are we facing?</a></li>
+
                         <li><a href="#section1">Expression Burden</a></li>
−
                         <li><a href="#section2">Predecessors</a></li>
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                         <li><a href="#section2">Capacity Monitor</a></li>
−
                         <li><a href="#section3">Project Xscape</a></li>
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                         <li><a href="#section3">VioABDE</a></li>
−
                         <li><a href="#section4">For Fermentation</a></li>
+
                         <li><a href="#section4">Alternative Device</a></li>
−
                         <li><a href="#section5">For Therapy</a></li>
+
                         <li><a href="#section5">References</a></li>
−
                        <li><a href="#section6">Metabolic Stress</a></li>
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                        <li><a href="#section7">DIY Bio and Biosafety</a></li>
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                        <li><a href="#section8">Community and Future</a></li>
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                        <li><a href="#section9">References</a></li>
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            <h1>Measurement</h1>
 
             <div class="topic-title" id="section1">
 
             <div class="topic-title" id="section1">
−
                 <h3>What are we facing?</h3>
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                 <h3>Expression Burden</h3>
−
                 <p>Biosafety has always been the major concern to the public, to the companies and the researchers. Doubts and worries raised just as genetic technology was invented. With the rapidly growing of synthetic biology and iGEM community, more and more synthetic biology products are built with the widely distributed DNA toolkits or the inexpensive DNA synthesis service(Synthetic and Will); we are facing unprecedented biosafety issue that unwanted leakage of synthetic biology products to the environment may cause an unexpected but definitely disastrous problem. </p>
+
                 <p>In synthetic biology, a common issue in integrating exogenous sequences into host cells is that heterologous gene expression can cause significant metabolic burden. Typically, this gene expression burden is implied by a declining growth rate of bacteria, which is unfavorable for fermentation industry because it lowers yields and profits. According to Ceroni et al. (2015), a fluorescence-based method allows to measure gene expression capacity of cells, by inserting a “capacity monitor”.</p>
 
             </div>
 
             </div>
 
             <div class="topic-title" id="section2">
 
             <div class="topic-title" id="section2">
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                 <h3>Predecessors</h3>
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                 <h3>Capacity Monitor</h3>
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                 <p>For decades, researchers were striving to build biosafety devices through auxotrophy or external inducive kill switches(Lee et al.), holins and restriction enzymes are most commonly used. Most of the failures of the previous devices were caused by mutation and evolution of immune(Moe-Behrens et al.) . </p>
+
                 <p>The “Capacity Monitor” consists of a superfold GFP with a strong constitutive promoter, which is constantly expressed in the bacterial genome. Thus, any alternation in fluorescence intensity can reflect in overall resource availability in the cell. Particularly, with expression of synthesized genes, the expression of GFP will decrease, thus reporting a reallocation of cellular resources. </p>
−
                <p>The two major threats of engineered microbes’ leakage are the possible Horizontal Gene Transfer which will lead to the spread of recombinant DNA to the entire ecosystem, or the engineered bacteria could contaminate or overrun the natural habitat.(Wright et al.)</p>
+
 
             </div>
 
             </div>
 
             <div class="topic-title" id="section3">
 
             <div class="topic-title" id="section3">
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                 <h3>Project Xscape</h3>
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                 <h3>VioABDE</h3>
−
                 <p>Under this circumstance, this year we decided to be a fundamentalist to synthetic biology, by using genetic circuits and logic gates, to establish biosafety devices which can apply to the real-world situation.</p>
+
                 <p>After applying capacity monitor, we compared the fluorescence of E. coli with an empty vector and E. coli with VioABDE inserted and expressed. Both strains were incubated for 24 hours with fluorescence and OD constantly monitored. However, the OD of bacteria with VioABDE increased faster than the bacteria without Vio expression, which contradicts our initial expectation.</p>
−
                <p>Since cell death and lysis mean there is a continual presence of free DNA in the environment, holins, which are most widely used are excluded from our choices, and colicin E2 nucleases (Darmstadt iGEM2016) came into our site. We choose site non-specific nucleases since the entire genome and plasmids needed to be entirely digested to prevent the spread, and we use nucleases from a different family to prevent the possible evolution of nuclease inhibitors. Artificial DNA, RNA, and amino acids are a good solution, but due to its high cost so far, it is not applicable to most of the user.</p>
+
  
 
             </div>
 
             </div>
 
             <div class="topic-title" id="section4">
 
             <div class="topic-title" id="section4">
−
                 <h3>For fermentation</h3>
+
                 <h3>Alternative Device</h3>
−
                 <p>The first device we build is for the fermentation; we want to execute the escaped engineered bacteria from the fermenter, accidentally or intentionally. We used two environment factors to monitor the bacteria’s situation: temperature and population density, they are both high and tunable in the fermenter. So, the device will initiate when temperature and density are both low. We used thermal sensitive regulator (NUS iGEM2017)(Piraner et al.) and quorum sensing regulator (MIT iGEM2004) (Canton et al.)as our sensor, sRNA(Storz et al.) and tetR family repressor PhlF(Glasgow iGEM2015)(Stanton et al.) as the signal inverter. We add intergrase (Peking iGEM2017) controlled by the thermal sensitive regulator, which will turn the promoter of a lethal gene when temperature rise in the fermenter so that bacteria can survive at the very beginning. Also, we build a model to stimulate the minimum autoinducer required at the beginning of the fermentation, same as the purpose of integrase. This model is for keeping bacteria alive at the very beginning of fermentation. Together they form a NOR gate which will lead to cell death through genome degradation when temperature and density decrease.</p>
+
                 <p>Since we target to modify bacteria used in fermentation industry, which primarily are used for material synthesis and production, our sequence inserts should occupy as limited resources in bacteria as possible to avoid significant expression burden. In order to reduce this expression stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking-S, 2011) and the “Cold Box” in 5’UTR region of CspA (Ionis Paris, 2017). With only one transcriptional regulator, less energy will be consumed, which is beneficial for fermentation industry.</p>
 
             </div>
 
             </div>
−
            <div class="topic-title" id="section5">
 
−
                <h3>For Therapy</h3>
 
−
                <p>The second device we build is for therapeutic bacteria, the device can carry out noninvasive tracing through ultrasound imaging of the gas vesicle(Shapiro et al.), release the drug (from SHSBNU 2017) controlled by a thermal sensitive regulator at nidus by ultrasound tissue heating, and heat to a higher temperature to release nuclease and kill the bacteria after it finishes its mission. </p>
 
−
            </div>
 
−
            <div class="topic-title" id="section6">
 
−
                <h3>For Metabolic Stress</h3>
 
−
                <p>We applied capacity monitor (Ceroni et al.) to quantify the expression burden of all our systems, and to reduce the metabolic stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking iGEM2011) and cold-regulated 5’UTR region (Ionis Paris 2017). This device only involves one transcriptional regulator, which will be less energy consuming. </p>
 
−
            </div>
 
−
            <div class="topic-title" id="section7">
 
−
                <h3>DIY bio and Biosafey</h3>
 
−
                <p>Back to the growing and glowing synthetic biology community, despite the ones doing it on campus, more and more people are starting it at home, they call themselves Genehacker or DIY biologists. The lack of sufficient training and efficient surveillance will be a time bomb which we do know there will be a monstrous harmful bioproduct will be made someday in the future, and indeed, it will be a significant threat to the current biosafety basis. Recall our memory to iGEM2009, Peking surveyed DIY bio, almost ten years later, we conducted a similar DIY bio-survey again. We tried to order materials for molecular experiments, using the delivery address to our home, the result was quite shocking that we can buy almost everything for the molecular experiment, from the internet. Then, we went through relevant laws and regulations throughout the world, which we found out that there are no laws related to the credit certification and the address certification about the people who book the biology reagent. Most of the laws are about the quality certification and how they would serve the user after they bought this. We interviewed the Director of the center for disease control and prevention. He said that within his experiment with the disease caused by the Bacteria leak, environmental pollution, the vast impact had been caused. Our country has been making all effort which is the highest effort that we have made in the history. He said it is not easy to solve the problem with hard work, it needs the cooperation between all the countries. He made an example of 731 army during the second world war two, the outbreak of pathogens can cause significant social harm. We are still on our way to win the battle, but the effort still needs to be put in.</p>
 
−
            </div>
 
−
            <div class="topic-title" id="section8">
 
−
                <h3>Community and Future</h3>
 
−
                <p>Also, we hosted two major meeting in Beijing, a Biosafety Forum in October, we invited team leader who runs his high school lab, lab teacher from a university lab, and a former team member from Peking iGEM2009, who participated in that DIY bio investigation ten years ago.</p>
 
−
                <p>We concluded that the development of DIY bio should be taken seriously, and the permanent way to solve it is through implanting Biosafety awareness into our academic culture. Also, as iGEMer, we should strive to be the considerable and responsible leaders in our community, to ensure the biosafety issue has been taken properly. Another meeting was with biology Olympians all around China, we discussed the future of biology community during the meeting, especially with more and more high school iGEM teams coming up in China, but lack of relevant instruction and education to the students. We came up with the idea of setting up a collaboration between school to share and overcome difficulties hand in hand. This kind of meeting will be continued after iGEM2018, since the community usually grows fast after every iGEM season. </p>
 
−
                <p>Hopefully, years later, biosafety awareness and considerations can be seriously taken in communities, laboratory studies, and real-world applications.</p>
 
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             <div class="topic-title" id="section5">
 
                 <h3>References</h3>
 
                 <h3>References</h3>
−
                 <p>Canton, Barry, et al. “Refinement and Standardization of Synthetic Biological Parts and Devices.” Nature Biotechnology, vol. 26, no. 7, 2008, pp. 787–93, doi:10.1038/nbt1413.</p>
+
                 <p>Ceroni, F., Algar, R., Stan, G. B., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature methods, 12(5), 415.</p>
−
                <p>Ceroni, Francesca, et al. “Quantifying Cellular Capacity Identifies Gene Expression Designs with Reduced Burden.” Nature Methods, vol. 12, no. 5, 2015, pp. 415–18, doi:10.1038/nmeth.3339.</p>
+
                 <p>Ionis Paris. (2017). Biological thermo-responsive solution, that reduces the plant’s thermal stresses. Available from: https://2017.igem.org/Team:IONIS-PARIS.</p>
−
                 <p>Lee, Jeong Wook, et al. “Next-Generation Biocontainment Systems for Engineered Organisms.” Nature Chemical Biology, Springer US, 2018, p. 1, doi:10.1038/s41589-018-0056-x.</p>
+
                 <p>Peking-S iGEM. (2011). A Chemical Wire Toolbox for Synthetic Microbial Consortia. Avaible from: https://2011.igem.org/Team:Peking_S.</p>
−
                <p>Moe-Behrens, Gerd H. G., et al. “Preparing Synthetic Biology for the World.” Frontiers in Microbiology, vol. 4, no. JAN, 2013, pp. 1–10, doi:10.3389/fmicb.2013.00005.</p>
+
−
                <p>Piraner, Dan I., et al. “Tunable Thermal Bioswitches for in Vivo Control of Microbial Therapeutics.” Food, Pharmaceutical and Bioengineering Division 2017 - Core Programming Area at the 2017 AIChE Annual Meeting, vol. 2, no. November, Nature Publishing Group, 2017, pp. 695–702, doi:10.1038/nchembio.2233.</p>
+
−
                <p>Shapiro, Mikhail G., et al. “Biogenic Gas Nanostructures as Ultrasonic Molecular Reporters.” Nature Nanotechnology, vol. 9, no. 4, Nature Publishing Group, 2014, pp. 311–16, doi:10.1038/nnano.2014.32.</p>
+
−
                 <p>Stanton, Brynne C., et al. “Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates.” Nature Chemical Biology, vol. 10, no. 2, 2014, pp. 99–105, doi:10.1038/nchembio.1411.</p>
+
−
                <p>Storz, Gisela, et al. “Regulation by Small RNAs in Bacteria: Expanding Frontiers.” Molecular Cell, vol. 43, no. 6, 2011, pp. 880–91, doi:10.1016/j.molcel.2011.08.022.</p>
+
−
                <p>Synthetic, How, and Biology Will. “Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves.” Choice Reviews Online, 2013, doi:10.5860/CHOICE.50-3835.</p>
+
−
                <p>Wright, Oliver, et al. “Building-in Biosafety for Synthetic Biology.” Microbiology (United Kingdom), vol. 159, no. PART7, 2013, pp. 1021–35, doi:10.1099/mic.0.066308-0.</p>
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Revision as of 14:00, 17 October 2018

Measurement

Expression Burden

In synthetic biology, a common issue in integrating exogenous sequences into host cells is that heterologous gene expression can cause significant metabolic burden. Typically, this gene expression burden is implied by a declining growth rate of bacteria, which is unfavorable for fermentation industry because it lowers yields and profits. According to Ceroni et al. (2015), a fluorescence-based method allows to measure gene expression capacity of cells, by inserting a “capacity monitor”.

Capacity Monitor

The “Capacity Monitor” consists of a superfold GFP with a strong constitutive promoter, which is constantly expressed in the bacterial genome. Thus, any alternation in fluorescence intensity can reflect in overall resource availability in the cell. Particularly, with expression of synthesized genes, the expression of GFP will decrease, thus reporting a reallocation of cellular resources.

VioABDE

After applying capacity monitor, we compared the fluorescence of E. coli with an empty vector and E. coli with VioABDE inserted and expressed. Both strains were incubated for 24 hours with fluorescence and OD constantly monitored. However, the OD of bacteria with VioABDE increased faster than the bacteria without Vio expression, which contradicts our initial expectation.

Alternative Device

Since we target to modify bacteria used in fermentation industry, which primarily are used for material synthesis and production, our sequence inserts should occupy as limited resources in bacteria as possible to avoid significant expression burden. In order to reduce this expression stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking-S, 2011) and the “Cold Box” in 5’UTR region of CspA (Ionis Paris, 2017). With only one transcriptional regulator, less energy will be consumed, which is beneficial for fermentation industry.

References

Ceroni, F., Algar, R., Stan, G. B., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature methods, 12(5), 415.

Ionis Paris. (2017). Biological thermo-responsive solution, that reduces the plant’s thermal stresses. Available from: https://2017.igem.org/Team:IONIS-PARIS.

Peking-S iGEM. (2011). A Chemical Wire Toolbox for Synthetic Microbial Consortia. Avaible from: https://2011.igem.org/Team:Peking_S.

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