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Venusian!!
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Abstract
 
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Are there Venusian? Is it possible that there are creatures living on the Venus? Dreams will come true by the symbiosis of cyanobacteria and eukaryotes.
 
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Cyanobacteria obtain their energy by photosynthesis. Sulfide-dependent photosynthesis cyanobacteria could resist the harmful environment and do the electron transport by sulfide quinine reductase(SQR), which we cloned previously. Additionally, references revealed when engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes, S. elongates was able to invade cultured mammalian cells.
 
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The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated. Results showed that invasin(inv,from Yersinia pestis) and listeriolysin O(llo, from Listeria monocytogenes) transformation would raise the entry speed and escape rate in J774 mouse macrophage cell line, and would increase the recruitment of cyanobacteria on induced pluripotent stem cells.
 
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Introduction
 
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Cyanobacteria obtain their energy by photosynthesis. In previous research in our study, we hope to create new type Synechococcus elongatus PCC7942 which could do both the nitrogen and sulfide metabolism. This new cyanobacteria would survive on the SO2-rich environment on the Venus. Meanwhile, when engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes (both provided by Pamela A. Silver, Harvard Medical school), S. elongates was able to invade cultured mammalian cells.
 
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    J774 mouse macrophage cell line, a kind of differentiate cells, could engulf cyanobacteria not only by phagocytosis but speedy by the inv-llo engineering help. Additionally, stem cells can divide and differentiate into diverse specialized cell types and can self-renew to produce more stem cells.  In a developing embryo, stem cells can differentiate into all the specialized cells (called pluripotent cells). Induced pluripotent stem cells (iPS cells ) are stem cell-like cells derived from reprogrammed somatic cells. IPS cells are then seeded on mouse embryonic fibroblast (MEF) feeder cells and cultured in ES cell medium. After separation iPS cells from MEF, non-EB iPS cells can be gotten. After culturing non-EB iPS cells on the petri dish, iPS cells at Embryoid Body(EB) stage can be obtained. Embryoid Body (EB) culture is used to examine the differentiation potential of the embryonic stem (ES) cells.
 
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Thus, if engineered cyanobacteria can invade into stem cells and produce energy and O2 by photosynthesis, a dream of creating Venusians would not be far.
 
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Methods and Materials
 
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What’s the specialty of our cyanobacteria?
 
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Invasin(protein names: P11922  INVA_YERPS) is a protein that allows enteric bacteria to penetrate cultured mammalian cells. Listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility[2]. And Synechococcus elongatus PCC 7942 was the first cyanobacterial strain which could be transformed by exogenously added DNA. When engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes (both provided by Pamela A. Silver, Harvard Medical school[3]), new S. elongatus was able to invade cultured mammalian cells and was capable of symbiosis with eukaryotic cells.
 
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    Additionally, invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes have been studied by 2010 Warsaw igem team. Our further study and novel observation relative to their research were posted not only on our wiki, but also on the partregistry website(BBa_K299810, BBa_K299811,BBa_K299812)
 
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What’s the platform of coculturing?
 
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Three types of mammalian cells were chosen—two stages of induced pluripotent stem cells from mice and J774 mouse macrophage cell line. Here are the protocols of cell culturing (two stages of mice induced pluripotent stem cells: iPS cells separated from embryonic fibroblasts (MEFs) click here, embryoid body (EB) stage of iPS cells click here, J774 mouse macrophage cell line click here)
 
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Cells were seeded on the 24-well plate. After 80% full in each well,  PCC7942(wt/inv-llo transform) suspensions in PBS were set to the same OD and 50 µl of this suspension were added per 1 ml of Leibovitz's L-15 medium without phenol red, without antibiotics and with 10% fetal bovine serum (Invitrogen) per well of 24-well tissue culture dishes containing the mammalian cells. After 24-hour cells and PCC7942 coculturing under 30°C atmospheric CO2 level, supernatants were discarded and cells were washed with PBS two times and the medium was replaced by L-15 containing antibiotics, gentamycin 100ug/ml(Invitrogen). After 24 hours, cells were fixed by 4% paraformaldehyde and were observed by fluorescent microscope or confocal laser microscope.
 
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Results and Discussion
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    There were three different types of cells we chose to do the coculture experiment. Part 1 revealed that J774 macrophage cell line enabled the entry of cyanobacteria by phagocytosis. Part2 and part3 were both mouse induced pluripotent stem cells but in different stage of stem cells. Secondly was non-EB iPS cells, separated from mouse embryonic fibroblasts (MEFs) and subcultured on culture dish. Thirdly was iPS cells in the Embryoid body (EB) stage. Later experiments showed three parts of results, J774 macrophage cell line(figure 2), non-EB iPS cells(figure 3), and iPS cells at the EB stage(figure 4). Red pots were S. elongates PCC7942 under fluorescence microscope 530-550 nm excitation filter. Under the transmitted light, transparent one was cells.
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Part 1
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    Figure 2 revealed coculturing results of J774 macrophage cell lines and S. elongatus PCC 7942(wildtype/inv-llo). After 24-hour coculturing and 24-hour cell culture under gentamycin 100 ug/ml, same concentration of wildtype and inv-llo transformed type(both were OD 0.05) both entered into J774 macrophage cell line by phagocytosis and the help of inv-llo.
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(A)inv-llo strain, merged image of fluorescence and bright field
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Figure 2. Red pots were S. elongates PCC7942.  Round and transparent one was J774 macrophage cell line. By phagocytosis, wildtype and inv-llo type entered into cells.
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In figure 2,(B)(D) were the images under the fluorescence microscope 530-550 nm excitation filter. Red pots were S. elongates PCC7942. Under the transmitted light, round and transparent one was J774 macrophage cell line. (A)(C) were the merged images of bright fields and fluorescence. Compared inv-llo transformed type to wildtype, red pots of inv-llo transformed type were less bright than those of wildtype. Additionally, inv-llo transformed type numbers in cells were less than wildtype numbers. It might suggest that inv-llo gene could raise the entry speed and the escape rate.
 
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Part 2
 
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Figure 3 showed the coculture results of non-EB iPS cells. Cells and S.elongatus PCC7942(wildtype/inv-llo). Images(A)(B)(E)(F)(G)(H) were inv-llo strain and non-EB iPS cells coculturing results (A,B: under fluorescence microscope E~H: under confocal laser microscope.) Cells and cyanobacteria at the separate focus revealed the negative symbiosis. However, inv-llo plasmid showed that inv-llo would recruit cyanobacteria to cells. 3D image under the confocal laser microscope(image H) revealed the recruitment of cyanobacteria.
 
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(A) inv-llo strain, merged image of fluorescence and bright field
 
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(B) inv-llo strain, image of fluorescence
 
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(C) wildtype, merged image of fluorescence and bright field
 
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(D) wildtype, image of fluorescence
 
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(E) inv-llo strain, merged image of confocal microscope and bright field, 1000X
 
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(F) inv-llo strain, image of confocal microscope, 1000X
 
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(G) inv-llo strain, image of bright field, 1000X
 
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(H) inv-llo strain, 3D image under confocal microscope, 1000X
 
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Figure 3. Red pots were S. elongates PCC7942 under fluorescence microscope.  Transparent one was non-EB iPS cells. Both wildtype and inv-llo type could not enter into cells but inv-llo type could trigger itself recruitment to cells.
 
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<div class="title">Becoming Venusian !</div>
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  <p><span class="subtitle">Overview</span></p>
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  <p>Are there <em><u>Venusian</u></em>? Is it possible that  there are creatures living on the Venus? Dreams will come true by the symbiosis  of cyanobacteria and eukaryotes.<br />
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    </p>
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  <p>Cyanobacteria obtain  their energy by photosynthesis. Sulfide-dependent photosynthesis cyanobacteria  could resist the harmful environment and do the electron transport by sulfide quinine  reductase(SQR), which we cloned previously. Additionally, references revealedwhen  engineered with invasin(inv) from <em>Yersinia</em><em>pestis</em> and  listeriolysin O(llo) from <em>Listeria monocytogenes</em>, <em>S. elongates</em> was  able to invade cultured mammalian cells.<br />
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  <p>The symbiosis possibility of induced pluripotent stem cells from mice  and J774 mouse macrophage cell line were evaluated. Results  showed that invasin(<em>inv</em>,from <em>Yersinia</em><em>pestis</em>) and listeriolysin O(llo, from <em>Listeria monocytogenes</em>) transformation would raise the entry speed and escape rate in J774 mouse macrophage cell line, and would increase the recruitment of  cyanobacteria on induced pluripotent stem cells. </p>
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            <h2 class="drawer-handle open">Denitrifying Machine</h2>
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                <li><a title="Background" href="http://2012.igem.org/Team:NYMU-Taipei/ymin1.html">Background</a></li>
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Part 3
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Figure 4 revealed the coculture results of S.elongatus PCC 7942 (wt/inv-llo strain)and iPS cells at EB stage.
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            <h2 class="drawer-handle">Sulfur Oxide Terminator</h2>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymis1.html">Overview</a></li>
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                <li><a title="Discussion" href="http://2012.igem.org/Team:NYMU-Taipei/ymis6.html">Discussion</a></li>
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                <li><a title="Conclusion & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymis7.html">Conclusion &amp; References</a></li>
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                <li><a title="Abstract" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq1.html">Abstract</a></li>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymic1.html">Overview</a></li>
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                <li><a title="Experiment Design" href="http://2012.igem.org/Team:NYMU-Taipei/ymic2.html">Experiment Design</a></li>
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                <li><a title="Results & Discussion" href="http://2012.igem.org/Team:NYMU-Taipei/ymic4.html">Results & Discussion</a></li>
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            <h2 class="drawer-handle">Becoming Venusian</h2>
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                <li><a title="Overview" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusian.html">Overview</a></li>
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                <li><a title="Introduction" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusianintro.html">Introduction</a></li>
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                <li><a title="Methods & Materials" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusianmm.html">Methods &amp; Materials</a></li>
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                <li><a title="Result & Discussion" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusianrd.html">Result &amp; Discussion</a></li>
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                <li><a title="Conclusion & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymivenusiancr.html">Conclusion &amp; References</a></li>
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(A) iPS cells at EB stage, image of bright field
 
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(B)inv-llo strain, image of fluorescence
 
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(C)inv-llo strain, merged image of fluorescence and bright field Figure 4.
 
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Red pots were S. elongates PCC7942 under fluorescence microscope. Gray one was iPS cells at the EB stage. Results showed that not many inv-llo PCC 7942 could enter into cells.
 
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There were similar and relative results of the iPS cells at EB stage and non-EB iPS cells. Both inv-llo type of S.elongatus PCC7942 didn’t enter into stem cells but triggered the recruitment to cells. Because embryoid body contained not only one cell, the entry for cyanobacteria would be more difficult but the recruitment provided a potential for invading mammalian cells. Future study will focus on different coculturing condition and after invasion into stem cells, differentiation could render Venusian.
 
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Conclusion
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Inv-llo cyanobacteria coculturing with induced pluripotent stem cells from mice, and J774 mouse macrophage cell line separately suggest the symbiosis possibility by the recruitment of cyanobacteria and the faster entry and escape rate.
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References
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1. The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes, Stephen Vadia1, Eusondia Arnett1, Anne-Cécile Haghighat1, Elisabeth M. Wilson-Kubalek2, Rodney K. Tweten3, Stephanie Seveau1*, PLoS One. 2011; 7(11): e1002356
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2. Towards a Synthetic Chloroplast, Christina M. Agapakis,1 Henrike Niederholtmeyer,1,¤ Ramil R. Noche,1 Tami D. Lieberman,1 Sean G. Megason,1 Jeffrey C. Way,2 and Pamela A. Silver1,2,PLoS One. 2011; 6(4): e18877.
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Revision as of 14:12, 17 October 2012

NYMU iGEM

Becoming Venusian !

Overview

Are there Venusian? Is it possible that there are creatures living on the Venus? Dreams will come true by the symbiosis of cyanobacteria and eukaryotes.

Cyanobacteria obtain their energy by photosynthesis. Sulfide-dependent photosynthesis cyanobacteria could resist the harmful environment and do the electron transport by sulfide quinine reductase(SQR), which we cloned previously. Additionally, references revealedwhen engineered with invasin(inv) from Yersiniapestis and listeriolysin O(llo) from Listeria monocytogenes, S. elongates was able to invade cultured mammalian cells.

The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated. Results showed that invasin(inv,from Yersiniapestis) and listeriolysin O(llo, from Listeria monocytogenes) transformation would raise the entry speed and escape rate in J774 mouse macrophage cell line, and would increase the recruitment of cyanobacteria on induced pluripotent stem cells.