Difference between revisions of "Team:Marburg"

 
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<h3>Accelerate engineering of biological systems by utilizing lab automation and state of the art lab technology</h3>
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There was a time, when building cars relied on delivering each frame to the workers for continuing the manufacturing process by horse-drawn carriages. In order to make car production more efficient, Henry Ford invented the first moving assembly line in industry, which is a fundamental part of car manufacturing all over the world today. In synthetic biology engineering principles like standardization, decoupling and abstraction enable the construction of biological system with new, non-natural functions
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<a href="http://icampus.mit.edu/files/2011/11/iGem-Nature-Review-Foundns.pdf"><abbr title="(Endy:  Foundations for engineering biology, Nature , (2005); 438(7067), pp. 449–453">(Endy 2005) </abbr></a>
 
  
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with applications in biomedical
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<a href="https://is.muni.cz/el/1431/podzim2014/Bi7430/um/50790219/Paddon_2014_review_NatRew_Artemisinin_story_as_model_for_SB_in_pharmaceutical_development__1_.pdf?lang=en"><abbr title="(Paddon and Keasling: Semi-synthetic artemisinin. A model for the use of synthetic biology in pharmaceutical development, Nature reviews. Microbiology, (2014); 12(5), pp. 355–367">(Paddon and Keasling 2014) </abbr></a>
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<a href="http://www.pnas.org/content/early/2018/03/27/1721469115"><abbr title="(Li, Yanran; Li, Sijin; Thodey, Kate; Trenchard, Isis; Cravens, Aaron; Smolke and Christina D.: Complete biosynthesis of noscapine and halogenated alkaloids in yeast., Proceedings of the National Academy of Sciences of the United States of America, (2018); 115(17), , E3922-E3931">(Li et al. 2018) </abbr></a>
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, agriculture
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<a href="http://cshperspectives.cshlp.org/content/early/2017/02/27/cshperspect.a023887.abstract"><abbr title="(Boehm, Christian R.; Pollak, Bernardo; Purswani, Nuri; Patron, Nicola and Haseloff, Jim: Synthetic Botany, Cold Spring Harbor perspectives in biology, (2017); 9(7)">(Boehm et al. 2017) </abbr></a>
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and biotechnological industry
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<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490365/"><abbr title="(Brent Erickson, Janet E Nelson and Paul Winters:  Perspective on opportunities in industrial biotechnology in renewable chemicals, Biotechnology journal, (2012); 7(2), pp. 176–185">(Erickson et al. 2012) </abbr></a>
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. Nevertheless, several challenges, such as managing biological complexity, unreliable construction of synthetic biological systems, variation in the behavior and evolution of these systems have to be overcome to establish synthetic biology as an engineering discipline. But how could we overcome these engineering challenges? One possible solution would be to learn from past lessons, when engineering disciplines, which have changed our world tremendously, emerged from natural science like physics and chemistry. But could we adapt these ideas from e.g. mechanical engineering to synthetic biology in a useful way? One example from past lesson is the manufacturing of cars.</p>
 
  
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Establishing <i>Vibrio natriegens</i> as the new chassis organism for synthetic biology.<br>
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With a doubling time of 7 minutes, we were able to complete a full cloning cycle in less than 12 hours!
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We established three new strains derived from the original wildtype strain as chassis organisms for cloning, protein expression and protein interaction studies. Moreover, we got first evidence for a CRISPR/Cas9 mediated knockout.
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We created the Marburg Collection, a highly flexible golden-gate based cloning toolbox consisting of 123 individual parts. Our novel measurement workflow was applied to obtain highly reproducible data on the behavior of our parts in <i>V. natriegens</i>.
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We developed a workflow for accelerating metabolic engineering. As a proof of concept we established the first synthetic pathway in <i>Vibrio natriegens</i> to produce the platform chemical 3-hydroxypropionic acid.
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To collaborate with other teams and spread the word to other scientists on <i>Vibrio natriegens'</i> advantages we had teams from all over Europe conduct a growth curve or a InterLab inspired experiment.
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This Wiki is designed to be accessible for everyone!
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For this years Human Practices, iGEM Marburg decided to partner up with the BLISTA again. The BLISTA is the only Highschool for visually impaired students in Europe. As part of our project we (and other teams) designed a barrier-free website to take this step to remove barriers in communication!
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      <b>Figure 1:</b> Rise of the robots: The evolution of Ford's assembly line By Sheena McKenzie, for CNN, April 29, 2015.
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Since the invention of the first moving assembly line in 1913, automation and utilizing robots found their way into car factory&rsquo;s and have accelerated the process of cars production so that every 4 seconds a ford is built somewhere in the world.</p>
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What can we learn from building cars more efficient for making biology easier to engineer? When we started our project, we tried to identify the most time-consuming step in synthetic biology and came to the conclusion that the waiting time for the growth of the chassis is one of the biggest drawbacks, which has to be overcome to accelerate the design, build and test process for engineering biological systems. But if you would transfer that to our car production analogy, it would be the same as exchanging all workers but sticking to all old and slow machines in the factory without all the automation, which has taken place. Only the combination with robots and an assembly line for production has led to the highly improved efficiency. Inspired by this idea, we set our goal to combine our project of establishing the fastest organism in the world with state-of-the-art lab automation and lab technology and reached out to several companies. The first step was to find a PCR cycler with the fastest ramp rate (heating/cooling per second) and we came across an application note
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<a href="https://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=2ahUKEwj2wsqRkI7eAhXQ_qQKHR2wDCgQFjAAegQICBAC&url=https%3A%2F%2Fonline-shop.eppendorf.com.br%2Feshopdownload%2Fdownloadbykey%2F49263_40&usg=AOvVaw2S_DuYyUO7SLCEHu2wBB6R"><abbr title="(Nils Gerke and Arora Phang:  Comparative Run Time Evaluations of PCR Thermal Cyclers, Eppendorf AG, (2017); APPLICATION NOTE No. 274">(Nils Gerke and Arora Phang 2017) </abbr></a>
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where different PCR cyclers were compared in terms of runtime. Here it was reported that the Mastercycler X50s from Eppendorf has the fastest ramp rate of 10&deg;C/Sec and we contacted Eppendorf and luckily, they provided us the cycler for the time of this year&rsquo;s competition and we were able to test the fast performance for our PCR applications. Therefore, we established a protocol for fast PCR&acute;s in order to speed up the cloning process and develop a fast and cheap site directed mutagenesis (SDM) workflow. In our hands we were able to perform a complete SDM cloning (point mutation, deletion, insertion) in one day. Additionally, the Mastercycler X50s was utilized for high throughput transformation (in 96 well plates), which is critical for other cycler models due to slow ramp rates, which decrease transformation efficiency.</p>
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      <b>Figure 2:</b> Gel electrophoresis after PCR protocol optimization for 20 min and 16 min protocols.
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The next step for our accelerated synthetic biology assembly line was bringing in the automation. Therefore, we applied for a free Opentron and were lucky to be provided by a free robot. This allowed us to automate processes like inoculating culture to a defined OD, coping plates and preparing 96 well plate transformations. Additionally, we performed a complete run of the interlab study by utilizing the Opentron robot.</p>
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Our main achievement was the establishment of an automated golden gate cloning workflow, where we were able to build over 50 plasmids by combining our cloning software tool Click&rsquo;n&rsquo;Clone and the pipetting robot. Therefore, we programmed a software interface between our cloning tool and our pipetting scripts. This enables easy and fast plasmid construction, with an extremely high layer of abstraction, where the user does not have to consider lower abstractions levels, which enables more complex synthetic biological designs. When we build all these constructs with the Opentron, we realized that building huge construct libraries is still time consuming even with our automation approach. If we bring back the idea of automation in the car industry, the next step would be to establish a complete assembly pipeline for our workflow. That is why we went back to literature and found a paper on utilizing &nbsp;sound waves for DNA fabrication.</p>
+
  
−
<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490365/"><abbr title="(Kanigowska, Paulina; Shen, Yue; Zheng, Yijing; Rosser, Susan and Cai, Yizhi: Smart DNA Fabrication Using Sound Waves. Applying Acoustic Dispensing Technologies to Synthetic Biology, Journal of laboratory automation, (2016); 21(1), pp. 49–56">(Kanigowska et al. 2016) </abbr></a>
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They have shown several down scaled cloning methods, where the pipetting process is replaced by acoustic droplet liquid handling via Echo devices. Nanoliter-scale liquid droplets can be transferred with high precision and accuracy by utilizing acoustic droplet ejection technology. This method minimizes the costs of reagents and consumables, due to noncontact, tipless, low-volume nature of this liquid handling approach. They could successfully downscale PCRs and cloning methods, such as Golden Gate and Gibson assemblies to the nanoliter scale and at the same time increasing the assembly efficiency and decreasing the reagent cost by 20- to 100-fold.</p>
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        <figcaption><b>Figure 4:</b> Inner workings of the Echo.
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Our vision was to combine the Echo as well as the Opentron for exactly creating our own assembly line of automation. We again got in contact with Labcyte and we were very happy to get the chance to be able to integrate an Echo into our assembly line. This allowed us to scale up our construct library size significantly by establishing our own automation process. This includes utilizing the Echo for combinatorial golden gate assemblies, using the Marburg Collection and subsequently pipetting higher volume steps with the Opentron robot. All the following cloning steps can be accelerated by combing the high throughput automation with the fast-growing <em>Vibrio natriegens.</em> Before we build our project related plasmid libraries, we were able to demonstrate Gibson cloning, Golden gate assemblies with 8 parts, Aqua cloning (also site directed mutagenesis based on Aqua cloning) and natural transformation with our lab automation process by downscaling all reaction volumes, which has not been shown for Aqua cloning and natural transformation before. For the natural transformation it was surprisingly for us, especially because it has been reported that high amounts of DNA are needed for the uptake of the DNA. In addition to using the Echo for transferring DNA, we also successfully tested the transfer of competent cells. When we thought about the next bottleneck in our automation, we realized that after transformation all transformed have to be plated separately by hand, which makes it nearly impossible to increase the size of the plasmid library. So, we came up with the idea of plating in a high through put way via the Echo. We could demonstrate to accelerate and scale up the plating process by plating 384 spots, which could be formed by one correct clone, if the cell solution is diluted properly. This would</p>
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        <figcaption><b>Figure 5:</b> 384 Well plate of the Echo.
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enable to automate the next step for a fully automated cloning workflow. The next step would be the picking step, and we also had different ideas on that, as some efforts are done to build an open source colony picker by using the Opentron. Unfortunately, we were not able to realize automated colony picking, but this would be an important missing step for the future. To demonstrate the precision of the Echo for shooting nanoliter droplets, we came up with the idea of printing our team logo on an agar plate with Vibrio Natriegens, what turned out successfully.</p>
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        <figcaption> <b>Figure 6:</b> Vibrigens Logo plated with the Echo.
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When we started with the idea of getting an Echo for our automation pipeline, our expectations were high in terms of how fast the Echo is in transferring combinatorial liquid handling tasks. But when we saw it the first time with our own eyes, all of us were incredibly impressed and our expectations were completely exceeded. We set up a test run of nearly 1000 transitions steps and after 3 minutes the run was done and the 96 well plate was filled with 8 part- golden gate reactions.</p>
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After establishing automation for the key methods of our cloning process we designed our main high throughput experiment. For that we used our metabolic engineering project as a proof of concept for our automation pipeline by building up a library for our synthetic metabolic pathway, where the expression levels are fine tuned by vary different promotor and RBS strength. In order to create all the pipetting scripts for the Echo, we wrote our own software viaMatlab, due to the fact that it would be impossible to write a picking list (10 pipetting steps for each plasmid x 1446) by hand.&nbsp; Moreover, we were able to write an interface for our Click`n`Clone software, which enables easy plasmid construction by the Echo via a graphical user interface.</p>
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        <figcaption><b>Figure 7:</b> Graphical user interface for the Echo.
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In the end we successfully constructed a library of 1446 variants of our pathway (link metabolic design). Constructing a library of this size would not have been possible without using the Echo, which is to our knowledge the fastest method for combinatorial, low volume liquid handling, as it needed for molecular cloning applications. After setting up the picking list we prepared our complete Marburg toolbox on a 384-well plate as a source plate for the Echo by establishing protocols for 384 well plates for the Opentron. As the number of parts increased over the time of the project, it became more critical to utilize the Opentron for this step, in order to minimize pipetting mistakes, which would lead to severe consequences in the following cloning procedure, especially for pipetting 384 well plates.</p>
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        <figcaption><b>Figure 8:</b> Black 384 well plate.
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All in all, we were able to successfully establish an accelerated assembly line for cloning, by combining the fastest growing organism, <em>V. natriegens, </em>with state-of-the-art lab technology and lab automation by utilizing an Opentron pipetting robot and the Echo acoustic liquid handler. We strongly believe that learning from past lessons and adapting ideas from mechanical engineering to synthetic biology could be a solution to overcome the challenges and drawbacks in synthetic biology to make biology an engineering discipline, which does not have to lack behind physics and chemistry. This will definitely include automation lines as it has changed other fields and industries in the past and will hopefully accelerate synthetic biology in the next decades to tackle the biggest challenges the humanity faces.</p>
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We want to thank again Eppendorf, Opentron and Labcyte, which enabled us to up to build up our Labautomation project by providing us with their devices for the time of our project. We also have to thank again Dominik Zahr from Labcyte, who helped us during the complete time of our Echo experiments and gave us an insight into what can be done with this amazing acoustic dispenser.</p>
+
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<h3>References</h3>
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−
<p>Boehm, Christian R.; Pollak, Bernardo; Purswani, Nuri; Patron, Nicola; Haseloff, Jim (2017): Synthetic Botany. In <em>Cold Spring Harbor perspectives in biology </em>9 (7). DOI: 10.1101/cshperspect.a023887.</p>
 
−
<p>Endy, Drew (2005): Foundations for engineering biology. In <em>Nature </em>438 (7067), pp.&nbsp;449&ndash;453. DOI: 10.1038/nature04342.</p>
 
−
<p>Erickson, Brent; Nelson; Winters, Paul (2012): Perspective on opportunities in industrial biotechnology in renewable chemicals. In <em>Biotechnology journal </em>7 (2), pp.&nbsp;176&ndash;185. DOI: 10.1002/biot.201100069.</p>
 
−
<p>Kanigowska, Paulina; Shen, Yue; Zheng, Yijing; Rosser, Susan; Cai, Yizhi (2016): Smart DNA Fabrication Using Sound Waves. Applying Acoustic Dispensing Technologies to Synthetic Biology. In <em>Journal of laboratory automation </em>21 (1), pp.&nbsp;49&ndash;56. DOI: 10.1177/2211068215593754.</p>
 
−
<p>Li, Yanran; Li, Sijin; Thodey, Kate; Trenchard, Isis; Cravens, Aaron; Smolke, Christina D. (2018): Complete biosynthesis of noscapine and halogenated alkaloids in yeast. In <em>Proceedings of the National Academy of Sciences of the United States of America </em>115 (17), E3922-E3931. DOI: 10.1073/pnas.1721469115.</p>
 
−
<p>Paddon, Chris J.; Keasling, Jay D. (2014): Semi-synthetic artemisinin. A model for the use of synthetic biology in pharmaceutical development. In <em>Nature reviews. Microbiology </em>12 (5), pp.&nbsp;355&ndash;367. DOI: 10.1038/nrmicro3240.</p>
 
  
 +
<section class="bottomSection">
 +
<div class="sectionContent">
 +
<aside>
 +
<div class="medalBody">
 +
<div class="sectionH scrollFade" fadeTo="fadeIn"><h1>Medal Criteria</h1></div>
 +
<div class="medal_container scrollFade" fadeTo="fadeIn">
 +
<div class="medalButton" mContent="m_a">
 +
          <img src="https://static.igem.org/mediawiki/2018/4/41/T--Marburg--BronzeCriteria.png" alt="">
 +
</div>
 +
<div class="medalButton" mContent="m_b">
 +
          <img src="https://static.igem.org/mediawiki/2018/3/38/T--Marburg--SilverCriteria.png" alt="">
 +
</div>
 +
<div class="medalButton" mContent="m_c">
 +
          <img src="https://static.igem.org/mediawiki/2018/0/01/T--Marburg--GoldCriteria.png" alt="">
 +
</div>
 +
<div class="medalButton" mContent="m_d">
 +
          <img src="https://static.igem.org/mediawiki/2018/9/97/T--Marburg--CrystalCriteria.png" alt="">
 +
</div>
 +
</div>
 +
<div class="medal_content_container">
 +
<div id="m_a" class="medal_content" tabindex="0">
 +
<h1>Bronze</h1>
 +
We fulfilled all bronze medal criteria.
 +
</h2>
 +
Our team registered for iGEM, we had a challenging but great iGEM season and we are really looking forward to attend the Giant Jamboree.
 +
<h2>Deliverables</h2>
 +
We already finalized our Wiki and <a href="https://igem.org/2018_Judging_Form?id=2560">Judging Form</a>. We will hold our presentation at the Giant Jamboree in Boston and we expect exciting discussions at our poster.
 +
<h2>Attributions</h2>
 +
Our project was only possible with a great <a href="https://2018.igem.org/Team:Marburg/Team">team</a> and we also want to thank people, institutions and companies who helped with financial and infrastructural support or scientific advice, and we are grateful to honor them on our <a href="https://2018.igem.org/Team:Marburg/Attributions">attributions page</a>.
 +
<h2>Characterization</h2>
 +
We participated in the fifth <a href="https://2018.igem.org/Team:Marburg/InterLab">iGEM InterLab</a>  measurement study, submitted our data in time and we are happy that our results were accepted by the measurement committee. 
 +
</div>
 +
<div id="m_b" class="medal_content" tabindex="0">
 +
<h1>Silver</h1>
 +
We are certain that we completed all silver medal criteria
 +
<h2>Validated Part</h2> We submitted <a href="http://parts.igem.org/Part:BBa_K2560069">BBa_K2560069</a> as a new basic parts. This part fulfills all formal requirements and was submitted to the registry. <a href="http://parts.igem.org/Part:BBa_K2560069">BBa_K2560069</a> is a 5’ Connector which was newly designed by us. We validated its function by showing that it can reduce crosstalk from backbone features 14 fold.
 +
<h2><a href="https://2018.igem.org/Team:Marburg/Collaborations">Collaborations</a></h2>
 +
We strengthened the iGEM community in Germany by hosting the <a href="https://2018.igem.org/Team:Marburg/Collaborations">German iGEM Meetup</a> and we planned and realized the <a href="https://2018.igem.org/Team:Marburg/Collaborations">Vibrigens InterLab study</a> with eleven participating teams.
  
−
</article>
+
<h2><a href="https://2018.igem.org/Team:Marburg/Human_Practices">Human Practices</a></h2>
 +
We got into contact with the BLISTA as part of our local community to open our project for everyone and we achieved to present our project in an accessible manner for the visually impaired.
 +
</div>
 +
<div id="m_c" class="medal_content" tabindex="0">
 +
<h1>Gold</h1>
 +
We hope that we can convince you that we fulfilled all gold medal criteria.
 +
<h2><a href="https://2018.igem.org/Team:Marburg/Human_Practices">Integrated Human Practices</a></h2>
 +
We integrated valuable input from society, academia and industry into the design and execution of our project by starting a wiki collaboration and implementing the suggestions of stakeholders into the design of our strains.
 +
<h2><a href="https://2018.igem.org/Team:Marburg/Improve">Improve a previous part</a></h2>
 +
We improved <a href="http://parts.igem.org/Part:BBa_P10500">BBa_P10500</a> by replacing the dropout with a sfGFP cassette, resulting in <a href="http://parts.igem.org/Part:BBa_K2560002">BBa_K2560002</a>.
 +
 +
<h2><a href="https://2018.igem.org/Team:Marburg/Model">Model Your Project</a></h2>
 +
Using modeling we predicted the metabolic pathway that yields optimal 3HPA producting and conducted foundational work towards a novel pathway.
 +
 +
<h2><a href="https://2018.igem.org/Team:Marburg/Demonstrate">Demonstration of Your Work</a></h2>
 +
We succeeded in all major parts of our project. We established genome engineering methods, created a toolbox and showed production of 3-HPA.
 +
</div>
 +
<div id="m_d" class="medal_content" tabindex="0">
 +
<h1>Special Prizes</h1>
 +
We are proud about what we achieved and applied for the following special awards:<br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Human_Practices">Integrated Human Practices</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Public_Engagement">Education and Public Engagement</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Model">Model</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Measurement">Measurement</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Software">Software Tool</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Measurement">Measurement</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Basic_Part">Best New Basic Part</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Composite_Part">Best New Composite Part</a><br><br>
 +
<a href="https://2018.igem.org/Team:Marburg/Part_Collection">Best Part Collection</a>
 +
</div>
 +
                </div>
 +
</div>
 +
<br>
 +
<br>
 +
<div class="medal_table" tabindex="0">
 +
<table>
 +
<tr>
 +
<th>Results</th>
 +
<th>Achieved</th>
 +
</tr>
 +
<tr>
 +
<td class="scrollFade slideInRight" fadeTo="transformIn">Initially, we carried out foundational experiments to characterize the growth rate, pH, salt and antibiotic tolerance of <i>V. natriegens</i> and sequenced its genome.
 +
</td>
 +
<td><div class="checkmark"></div></td>
 +
</tr>
 +
<tr>
 +
<td class="scrollFade slideInRight" fadeTo="transformIn">We performed Cloning in One day: From transformation to isolated plasmids in less than 12 hours!</td>
 +
<td><div class="checkmark"></div></td>
 +
</tr>
 +
<tr>
 +
<td class="scrollFade slideInRight" fadeTo="transformIn">Low amounts of DNA could be transformed reliably and we successfully demonstrated challenging clonings such as Gibson Assembly with 5 and Aquacloning with 3 fragments as well as 8 part golden-gate reactions.</td>
 +
<td><div class="checkmark"></div></td>
 +
</tr>
 +
<tr>
 +
<td class="scrollFade slideInRight" fadeTo="transformIn">We designed and constructed the Marburg Collection, a highly flexible golden-gate based toolbox consisting of 123 parts.
 +
</td>
 +
<td><div class="checkmark"></div></td>
 +
</tr>
 +
<tr>
 +
<td class="scrollFade slideInRight" fadeTo="transformIn">Dozens of test constructs were built and tested to obtain characterization data for all part categories in our toolbox.
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<td class="scrollFade slideInRight" fadeTo="transformIn">This was only possible with our novel experimental and data analysis workflow using platereader measurements.
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<td class="scrollFade slideInRight" fadeTo="transformIn">We successfully demonstrated a genome engineering workflow in <i>V. natriegens</i> to establish three new lab strains for diverse applications.
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<td class="scrollFade slideInRight" fadeTo="transformIn"><div class="checkmark"></div></td>
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<td class="scrollFade slideInRight" fadeTo="transformIn">Subsequently, we were able to use Flp/FRT recombinase system for marker recycling thus allowing additional rounds of genomic modifications.</td>
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<td class="scrollFade slideInRight" fadeTo="transformIn">Chromosomal locations suitable for genomic integration were identified and characterized to detect possible fitness effects. </td>
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<td class="scrollFade slideInRight" fadeTo="transformIn">We accelerated metabolic engineering by developing a workflow for rapid pathway assembly and pathway optimization in <i>V. natriegens</i>.</td>
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<td class="scrollFade slideInRight" fadeTo="transformIn">Model-predicted parts were used to construct a pathway for maximal 3-hydroxypropionate production in <i>V. natriegens</i> and we demonstrated its functionality.</td>
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<td class="scrollFade slideInRight" fadeTo="transformIn">Via mass spectrometry, we detected our pathway product                          3-hydroxypropionic acid in <i>V. natriegens</i>.
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Latest revision as of 13:35, 3 December 2018

Grand Prize Winner
Awards
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Strain Engineering
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Metabolic Engineering
Interlab
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B. Marchal