Difference between revisions of "Team:UCopenhagen/HP/Gold Integrated"

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                         <h1> I N T E G R A T E D &ensp;  H U M A N &ensp; P R A C T I C E S</h1>
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                     <h2 class="section-heading">Introduction </h2>
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                     <h2 class="section-heading"> </h2>
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                     <p class="lead">Our team believes that establishing a stable platform for scientists to create naïve orthogonal living compartments, would allow for an unpredictable advancement in the field of synthetic biology. Our project will not attempt to create an endosymbiont, but instead investigate the mechanisms in free-living cells in a bottom-up approach to endosymbiosis. 
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The endosymbiotic theory, formulated in the early years of the previous century, outlines that the organelles of the eukaryotic cell, such as the mitochondria, have their origin in free-living prokaryotes engulfed by bigger cells. These incorporated cells then co-evolved with their host conferring to it novel emergent properties which ultimately helped fuel the development of more complex multicellular biological systems such as plants and animals (Archibald, 2015). </p>
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Incell’s human practices explore the ethical, legal, social, economic, safety and security components of our work. They enshroud the beginning, middle and projected future of our work and Incell would not be what it is without.
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<br><br>
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<strong>Our goal </strong> is to actively engage the scientific community and general public through diverse communications and activities to guarantee our project is safe, responsible and good for the world.
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<br><br>
 +
We include a summary of our progress so far, what we have learned and what we plan to do with it.<br><br>
  
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We have learned and grown in the direction of the insights, knowledge and perspective gleaned from the humans which have shone on us. This is not a recent behaviour. We were we born and raised in human practices too, which you can read more about <a href="https://2017.igem.org/Team:UCopenhagen/HP/Gold_Integrated">here</a>.
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<p>We have identified three mechanisms we believe to be mandatory for the development of a stable endosymbiotic relationship, which we will be trying to replicate in free-living cells. First of all, in order for the relationship to be stable, the two organisms must  be mutually dependent on each other; there must be a mutually beneficial interaction between host and symbiont. Secondly, there has to be some sort of control and synchronization of symbiont replication. If the symbiont were to be replicating freely we could end up with way too many or not enough symbionts in the host.  Finally, a common feature of the endosymbiotic organelles we have looked at, is the transfer of genes from the symbiont to the host. Because of this transfer, the gene and protein expression is taking place in the nucleus and the proteins and metabolites are transported to the organelle. This import of proteins is interesting not just for understanding endosymbiosis, but also for the potential applications in synthetic biology.</p>
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<p>Based on these considerations, we decided to work on three distinct, but intertwined, projects pertaining to endosymbiosis, namely Interdependence, Number Control, and Protein import. We believe that by combining these three projects, a key step towards the understanding of endosymbiosis and its employment in synthetic biology will be obtained. </p>
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We engaged the scientific community and the public, conjuring an incredible and reciprocated curiosity. Platforms including conferences, social media (Facebook, Twitter, Instagram and LinkedIn), the Incell podcast, public presentations and interactive events have been spun together to form a warm, wool-like, structural skeleton around the team — a feeling we see as Incell’s moral compass.<br><br>
  
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We are creative, targeted and context appropriate with all communications and outreach. We have ferociously adapted to what works and what does not. It is entirely the point, upon seeing the reflection of our work in the face of humanity, to be sincerely changed by it.
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                    <h2 class="section-heading">Applications and Implications</h2>
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                    <<p>By understanding the basic principles behind the creation of stable endosymbiotic events we hope that in the future it will be possible to use artificial endosymbiosis as a new technology in synthetic biology, and we believe that value can be created in the foundational track of the iGEM competition. History has shown that great scientific advances has followed the implementation of new revolutionary technologies (Gershon 2003). </p>
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<p>We envision that artificial endosymbiosis could be applied in a broad range of fields, including agriculture, medicine and production of valuable compounds. A deeper understanding of the relationships intertwining endosymbionts and their hosts could unravel new knowledge applicable for the treatment of mitochondrial diseases, while a living compartment able to fixate nitrogen from the air could decrease the fertilizer use in agricultural production. </p>
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<p>However, the applications are only limited by the imagination of future users. Indeed, the game-changing role of endosymbiosis has not gone unseen to the eyes of the modern bioengineers, who predict that the establishment of a novel interaction has the potential to radically alter the host cell physiology without directly affecting the host genome (Scientific America Vol 105 pp. 36-45).</p>
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<p>Before the potential application of artificial endosymbiosis, there are many things to consider. While the current regulations regarding GMO limits what is possible to apply in agriculture and medicine, regulations regarding synthetically modified organisms (SMOs) have not yet been systematically put into place. How will a new field of SMO be regulated, and how will it influence possible applications of artificial endosymbiosis?</p>
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<p>In addition to our scientific investigation we are enthused to trigger debate about synthetic biology. We intend to podcast intriguing conversations with experts, thereby hoping to reach the general public and impel the discussion about the ethics and future prospects in combining biology and engineering.</p>
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<p>For both the Interdependence and Number Control projects we have chosen to use Escherichia coli as our chassis. As for the Endocytosis, multiple bacteria spanning different taxonomical groups will be examined. In the following paragraphs, a concise experimental plan for each of the projects will be described.</p>
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The take home message: for many, synthetic biology is unfamiliar and unless careful with wording — frightening. Many drew from their knowledge and misinformation of GMOs, and seeing SMOs as an unchartered extension, allow their scepticism to peak. Even after safety fears and dystopian nightmares are quashed, a more complicated question remains. The philosopher Sune Holm, speaking at our recent public forum on ethics in synthetic biology, said, “from a moral perspective, the main challenge is the question of who actually benefits from the creation of SMOs?” <br><br>
  
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These notes aside, the overarching vibe from the public has been, and hopefully continues to be, genuine curiosity. Moreover, some excitedly begin to formulate possible functions and applications. This is hugely rewarding feedback and evidence for Incell’s value as a technology in development. <br><br>
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<h3>Interdependence</h3>
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<p>The goal of the interdependence project is to explore the use of amino acid auxotrophy as the framework for a mutually beneficial relationship between two different organisms, namely engineering bacteria to export essential nutrients in a readily available form and in the proper quantity to be sufficient for the host. This will create a strong dependency in a host lacking the production facility of these nutrients. We will perform the experiments on free-living cells, and investigate if the exported amino acids would be sufficient to sustain a S. cerevisiae host. </p>
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The scientific community were free of safety fears and less astonished by the scientific objectives for Incell’s first phase. Importantly, they were equally excited, if not more so, by the vision and opportunities all of a sudden imaginable.<br><br>
  
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<p> As part of the interdependence project the following will be examined:</p>
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It is exceptionally important and too often given too little weight in science: communication and public engagement. Creating, organising and participating in public events is what has made Incell real — before it ever was.
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<li> the functional expression of amino acid exporters in E. coli; </li>
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<li>the level of amino acid produced and exported by the symbiont;</li>
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<li> the effect of nutrient export on E. coli cell viability and growth; </li>
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<li> Grow auxotrophic S. cerevisiae in co-culture with respective amino acid exporting E. coli. </li>
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<li>In silico modelling growth of S. cerevisiae under the levels of amino acids that can be exported by E. coli to investigate the minimum number of E. coli symbionts to sustain an auxotrophic yeast host cell.</li>
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<h3>Number control</h3>
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<p>The goal of the number control project is to lower or stop symbiont replication in case of a high symbiont abundance and/or a starvation status of the host cell, with proof of concept being performed in E. coli cultures. To intertwine the cell replication cycle and lower the stress created by the presence of a symbiont inside the cytoplasm of the host, we aim to put the number control system under control with three signals: the symbiont abundance (a quorum sensing circuit), the host cell starvation status, and the host cell replication. QS and cell starvation should lower symbiont replication, while host replication should increase it.
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We aim to establish a replication control and intertwinement using a modular system based on CRISPR/Cas9 technology. A catalytically-dead Cas9 (dCas9) lacking endonuclease activity and a small guide RNA will be guided via RNA-DNA interaction to the origin of replication on the bacterial chromosome, to efficiently and transiently inhibit the chromosome replication (Wiktor J. et al, 2016).</p>
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<p>The dCas9 system will be put under control of the quorum sensing pathway with the lux promoter, which will be active during high symbiont density.
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Additionally, we aim to put the number control system under control of cell starvation status and host cell replication. Moreover, to overcome an unrestrained cell growth, we aim to inhibit the membrane production silencing the expression of a key enzyme for lipid biosynthesis, i.e. Enoyl Acyl Reductase, commonly target of bacteriostatic drugs.</p>
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<p> As part of the Number Control project the following will be examined:</p>
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<li> The effect of dCas9 expression on the cell cycle of E. coli </li>
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<li>The effect of sgRNA binding to various site of the chromosomal origin of replication  on the cell cycle of E. coli</li>
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<li>The effectiveness of the two parts, i.e. dCas9 and sgRNA, to block DNA replication and cell growth </li>
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<li> QS efficiency in controlling dCas9 expression</li>
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<li>Inhibition of Enoyl Acyl Reductase expression via dCas9 in combination with a sgRNA targeting the enzyme promoter.</li>
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<p>The integration of the host signals, i.e. the host starvation status and the cell cycle phase, to control the dCas9 expression will be tested using an in-silico model.</p>
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<h3>Protein import</h3>
 
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<p>The goal of the protein import project is to import fluorescent proteins into a bacteria by covalently connecting the protein to a cell-penetrating peptide (CPP).</p>
 
−
<p>CPPs are small peptides (down to 8 amino acids), generally rich in arginine molecules, capable of initiating cellular uptake of a large variety of molecules and proteins by inducing endocytosis. In both plant and mammalian cells CPPs have been shown to mediate protein uptake by both covalent and non-covalent association, with increased specificity during covalent association. However, in bacteria only non-covalent uptake of proteins have so far been demonstrated (Chang et al. 2014). We will explore the utilization of the synthetic CPP nona-arginine (R9) as a vector for facilitation of targeted protein import in bacteria.</p>
 
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<p> As part of the Endocytosis project the following will be examined: </p>
 
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<li> An initial screening process aimed at evaluating the ability of a variety of bacterial species to take up fluorescent proteins through covalent and non-covalent CPP association </li>
 
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<li>Investigation of the cellular localization of proteins taken up in a CPP-mediated fashion. </li>
 
−
<li>If proteins taken up are localized in membrane derived vesicles we will further investigate the potential ability of last 20 amino acids of influenza virus hemagglutinin (H2) to facilitate vesicular escape.  </li>
 
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<br>
 
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<p>Proteins needed for this sub-project will be expressed with His-tags in E. coli and purified using affinity chromatography. Both flow cytometry and confocal microscopy will be used to evaluate bacterial protein uptake through use of the fluorescent proteins YFP and BFP as previously described (Chang et al. 2014). A lipophillic dye capable of staining membrane-derived vesicles (e.g. FM4-64) will be used to investigate cellular localization. To facilitate cleavage of R9 following import, either an endogenous or heterologously expressed peptidase will be used. Successful cleavage will be assessed through use of proteomics (e.g. targeted proteomics). The exact details for evaluation of enzymatic ability following protein import have still not been elucidated, we are however currently contemplating using an enzyme whose activity can be monitored through a colorimetric assay (e.g. 𝜷-lactamase).</p>
 
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                <h2>References</h2>
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<li> Archibald, J. M. Endosymbiosis and Eukaryotic Cell Evolution. Current Biology 2015, 25: 911–921. </li>
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<li>Diane Gershon Technology: pushing the boundaries of scientific discovery. Nature Medicine 2003; 9:97</li>
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<li>Jakub Wiktor, Christian Lesterlin, David J. Sherratt, Cees Dekker; CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res 2016; 44 (8): 3801-3810.</li>
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<li>Microsugar Chang, Yue-Wern Huang, Robert S. Aronstam and Han-Jung Lee; Cellular Delivery of Noncovalently-Associated Macromolecules by Cell-Penetrating Peptides. Current Pharmaceutical Biotechnology 2014; 15: 267-275. </li>
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                     <h2>Find Incell here:</h2>
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Revision as of 00:15, 2 November 2017

I N T E G R A T E D   H U M A N   P R A C T I C E S

Incell’s human practices explore the ethical, legal, social, economic, safety and security components of our work. They enshroud the beginning, middle and projected future of our work and Incell would not be what it is without.

Our goal is to actively engage the scientific community and general public through diverse communications and activities to guarantee our project is safe, responsible and good for the world.

We include a summary of our progress so far, what we have learned and what we plan to do with it.

We have learned and grown in the direction of the insights, knowledge and perspective gleaned from the humans which have shone on us. This is not a recent behaviour. We were we born and raised in human practices too, which you can read more about here.

What have we done?

We engaged the scientific community and the public, conjuring an incredible and reciprocated curiosity. Platforms including conferences, social media (Facebook, Twitter, Instagram and LinkedIn), the Incell podcast, public presentations and interactive events have been spun together to form a warm, wool-like, structural skeleton around the team — a feeling we see as Incell’s moral compass.

We are creative, targeted and context appropriate with all communications and outreach. We have ferociously adapted to what works and what does not. It is entirely the point, upon seeing the reflection of our work in the face of humanity, to be sincerely changed by it.

What have we learned?

The take home message: for many, synthetic biology is unfamiliar and unless careful with wording — frightening. Many drew from their knowledge and misinformation of GMOs, and seeing SMOs as an unchartered extension, allow their scepticism to peak. Even after safety fears and dystopian nightmares are quashed, a more complicated question remains. The philosopher Sune Holm, speaking at our recent public forum on ethics in synthetic biology, said, “from a moral perspective, the main challenge is the question of who actually benefits from the creation of SMOs?”

These notes aside, the overarching vibe from the public has been, and hopefully continues to be, genuine curiosity. Moreover, some excitedly begin to formulate possible functions and applications. This is hugely rewarding feedback and evidence for Incell’s value as a technology in development.

The scientific community were free of safety fears and less astonished by the scientific objectives for Incell’s first phase. Importantly, they were equally excited, if not more so, by the vision and opportunities all of a sudden imaginable.

It is exceptionally important and too often given too little weight in science: communication and public engagement. Creating, organising and participating in public events is what has made Incell real — before it ever was.

Find Incell here: