Revision as of 23:18, 19 October 2016

Projects :: Syndustry - iGEM Marburg 2016

SynDustry Fuse. Use. Produce.

Fusion of different organisms using polyethylene glycol (PEG)

Different approaches to create artificial endosymbiosis have been described previously. The used methods vary depending on the host organism, including microinjection, invasion or phagocytosis for mammalian cells and a polyethylene glycol (PEG) -mediated fusion of vesicle enclosed microorganisms with yeast spheroplasts [1][2]. Even though the uptake was successful, these methods have not been utilized to date.

In order to realize our idea of a mostly self-sustainable production we had to decide for one an applicable uptake mechanism of the symbiont into the host organism. Using mainly S. cerevisiae and E. coli, we chose the aforementioned PEG-induced uptake due to its easy applicability for different yeast strains without further engineering.

Figure 1. Overview scheme for the single steps using the PEG mediated fusion protocol.

The procedure, which has been described before [2][3], has been adapted and optimized for regeneration of the S. cerevisiae cell wall in liquid media. After initial growth of S. cerevisiae host cells and desired endosymbionts to a mid-logarithmic culture, the cell wall of S. cerevisiae is digested using Zymolyase, an enzyme which cleaves the 1,3-ß-glucane bonds of the yeast’s cell wall [4]. Therefore, the yeast cells are only enclosed by their lipid membrane. Additionally, this step can be universally applied to most yeast strains since they do not vary much in cell wall components rather than in the ratio of these. The digestion of the cell wall is a necessary requirement for the next crucial step: enclosure of the chosen endosymbiont by PEG liposomes.

For the formation of liposomes under aqueous conditions, PEG of different molecular weights, ranging from 3,350 to 20,000 kDa has been used for E. coli. The molecular weight of used PEG can be adapted according to the size of the endosymbiont since as it correlates directly with the diameter of formed liposomes [5].

After mixture and gentle centrifugation of the cells, PEG can be added directly to the pellet. This step should be repeated several times with a dilution of the PEG concentration at every step. Next, the cells can be completely resuspended in a defined medium which enables the yeast cells to regenerate their cell wall. In order to select only those cells which have fused, a suitable selection pressure (for example a carbon source dependency for yeast and a low pH for E. coli) needs to be established.

Literature

[1] Agapakis, Christina M., et al. "Towards a synthetic chloroplast." PLoS One 6.4 (2011): e18877.
[2] Yamada, Takashi, and Kenji Sakaguchi. "Polyethylene glycol-induced uptake of bacteria into yeast protoplasts." Agricultural and Biological Chemistry 45.10 (1981): 2301-2309.
[3] Guerra-Tschuschke, I., I. Martin, and M. T. Gonzalez. "Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions." Applied and environmental microbiology 57.5 (1991): 1516-1522.
[4] Pastor, FI Javier, et al. "Structure of the Saccharomyces cerevisiae cell wall: mannoproteins released by zymolyase and their contribution to wall architecture." Biochimica et Biophysica Acta (BBA)-General Subjects 802.2 (1984): 292-300.
[5] Boni, Lawrence, et al. "Preparation of large liposomes by infusion into PEG." U.S. Patent Application No. 09/999,191.

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-             <img src="https://static.igem.org/mediawiki/2016/6/64/T--Marburg--SkizzePatrick_Skizzen_LS.svg"
+             <img src="https://static.igem.org/mediawiki/2016/2/2c/T--Marburg--SkizzePatrick_Skizzen_LS.svg"
                   class="img-responsive center-block figure_img" alt="Figure 1">
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              <p>
 The procedure, which has been described before [2][3], has been adapted and optimized for regeneration of the <i>S. cerevisiae</i> cell wall in liquid media. After initial growth of <i>S. cerevisiae</i> host cells and desired endosymbionts to a mid-logarithmic culture, the cell wall of <i>S. cerevisiae</i> is digested using Zymolyase, an enzyme which cleaves the 1,3-ß-glucane bonds of the yeast’s cell wall [4]. Therefore, the yeast cells are only enclosed by their lipid membrane.  Additionally, this step can be universally applied to most yeast strains since they do not vary much in cell wall components rather than in the ratio of these. The digestion of the cell wall is a necessary requirement for the next crucial step: enclosure of the chosen endosymbiont by PEG liposomes.
-            </p>
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-    </div>
-    <div class="row">
-        <div class="col-sm-12">
-            <img src="https://static.igem.org/mediawiki/2016/9/94/T--Marburg--Expression_plasmid_map_BP.jpeg"
-                 class="img-responsive center-block figure_img" alt="Figure 2">
-            <div class="figure_text">
-                <b>Figure 2. Plasmid map of the expression plasmid in <i>S. cerevisisae</i>.</b>
-                The plasmid consists of a bidirectional promoter region that regulates the expression
-                of the <i>mae1</i> gene and the <i>matB</i> gene. The fragments were inserted into the
-                pNK26 backbone that carries an ampicillin resistance for selection in <i>E. coli</i> and
-                a Trp1 cassette for selection in <i>S. cerevisiae</i>. The plasmid was assembled via gibson
-                assembly reaction.
-            </div>
-        </div>
-    </div>
-    <div class="row">
-        <div class="col-sm-12 text-justify">
-            <p>
-If malonate, which is essential for yeast viability and as it serves as fuel for the biosynthesis reaction is removed from the media it has to be delivered by the invading <i>E. coli</i> strain. To achieve this major changes in the beta-alanine route of <i>E. coli</i> had to be made to direct flux towards malonic acid production [3]. We designed an operon-plasmid consisting of six different genes from various organisms including genes from <i>E. coli</i>, which was overexpressed to increase the yield of produced malonic acid (Fig. 3). As backbone we used the part of the pACYC184 plasmid that consists of the <i>E. coli</i> origin p15A ori and the chloramphenicol resistance gene under its native cat promoter as the marker-gene. The operon itself was under the control of the constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23108">BBa_J23108</a> and consists of the <i>ppc</i> gene from <i>E. coli</i> that encodes for a phosphoenolpyruvate carboxylase that catalyzes the addition of bicarbonate to phosphoenolpyruvate (PEP) to form oxaloacetate [6], the <i>yneI</i> gene from <i>E. coli</i> that encodes for a succinic semialdehyde dehydrogenase that should catalyze the oxidation of malonic semialdehyde to malonic acid [3] and the <i>aspA</i> gene from <i>E. coli</i> that encodes for an aspartate ammonia lyase that catalyzes the reaction of fumaric acid to aspartic acid [7]. Additionally, we integrated the <i>panD</i> gene from <i>C. glutamicum</i>, which encodes for an aspartate-α-decarboxylase that catalyzes the reaction of aspartic acid to β-alanine [3] and the <i>pa0132</i> gene from <i>P. aeroginosa</i> that encodes for a β-alanine pyruvate transaminase that catalyzes the reaction of  β-alanine to malonic semialdehyde [3]. Last but not least, we integrated the <i>mae1</i> gene from <i>S. pombe</i> that encodes for a permease for malate and other C4 dicarboxylic acids [4], that should enable the strain to segregate malonic acid [5]. The first three genes of the operon (<i>panD</i>, <i>aspA</i> and <i>pa0132</i>) were regulated by the RBS <a href="http://parts.igem.org/Part:BBa_J61101">BBa_J61101 </a> with an related strengths of 22.7 %. The last three genes (<i>ppc</i>, <i>yneI</i> and <i>mae1</i>) were under the control of the RBS <a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032 </a> with a related strengths of 33.96 %. As terminator we used the double terminator <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a> with a forward efficiency of 0.984.
-            </p>
-        </div>
-    </div>
-    <div class="row">
-        <div class="col-sm-12">
-            <img src="https://static.igem.org/mediawiki/2016/2/29/T--Marburg--Operon_plasmid_map_BP.jpeg"
-                 class="img-responsive center-block figure_img" alt="Figure 2">
-            <div class="figure_text">
-<b>Figure 3. Plasmid map of the Operon plasmid in <i>E. coli</i>.</b>
-The plasmid consists of the BBa_B0015 terminator and the constitutive promoter BBa_J23108 that controls the expression of <i>panD</i>, encoding for an aspartate-α-decarboxylase, <i>aspA</i>, encoding for aspartase ammonia lyase and <i>pa0132</i>, encoding for a β-alanine pyruvate transaminase, regulated by the BBa_J61101 ribosome binding site. Additionally, <i>ppc</i>, encoding for a phosphoenolpyruvate carboxylase, <i>yneI</i>, encoding for a succinic semialdehyde dehydrogenase and <i>mae1</i>, encoding for a permease, regulated by the BBa_B0032 ribosome binding site. The operon was inserted into a pACYC184 backbone carrying a chloramphenicol resistance marker gene and a p15A <i>E. coli</i> origin.
-            </div>
-        </div>
-    </div>
-    <div class="row">
-        <div class="col-sm-12 text-justify">
-            <p>
-Both, the operon plasmid and the expression plasmid were assembled via Gibson assembly. <i>Ppc</i>, <i>aspA</i> and <i>yneI</i> were amplified using genomic DNA from the <i>E. coli</i> strain MG1655. <i>PanD</i>, <i>matB</i> and <i>pa0132</i> were amplified using synthesized nucleotide sequences as templates <i>Mae1</i> was amplified using genomic <i>S. pombe</i> DNA as template.
              </p>
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              <p>
-A different approach, to achieve the goal of reliably interacting organisms, was to set a protein-based dependency. In this scenario the yeast has an essential protein-coding gene knocked out, which then is complemented by <i>E. coli</i> expressing and secreting the protein. The targeted genes in yeast can vary from classic knockout genes such as <i>trp</i>, which can be supplemented from the medium to more unique knockouts; for example a subunit of the yeasts ribosome. The expression of the corresponding protein in <i>E. coli</i> should be regulated by an inducible promoter where the inducer has to be able to pass the yeast cell wall and membrane, such as T7 or <i>lac</i>. Additionally, this has the advantage that it can be easily controlled whether a survival of both cells is a result of the dependency or occurred for different reasons e.g. leaving out the induction.
+For the formation of liposomes under aqueous conditions, PEG of different molecular weights, ranging from 3,350 to 20,000 kDa has been used for <i>E. coli</i>. The molecular weight of used PEG can be adapted according to the size of the endosymbiont since as it correlates directly with the diameter of formed liposomes [5].
              </p>
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              <p>
-For the export we chose two different approaches. The first, was to construct a fusion protein consisting out of the protein of interest and YebF fused to its N-terminal end. YebF occurs in the genome of <i>E. coli</i> and has shown to be capable of exporting fused proteins due to unknown mechanisms [8]. The second approach was similar, yet a little bit more complicated. We fused the 178 bp signal sequence of the Flagellin encoding gene <i>fliC</i> upstream of the coding region of the desired protein. This sequence acts as a signal for the directed transport and assembly into the flagellum. A knockout of FliC and FliD interferes with the regulation of both transport and assembly. Hence, instead of Flagellin our protein of interest is transported directly to the membrane, where it gets secreted into the medium due to defective assembly. In addition, the FliC signal sequence has shown to be cleaved during this procedure [9].
+After mixture and gentle centrifugation of the cells, PEG can be added directly to the pellet. This step should be repeated several times with a dilution of the PEG concentration at every step. Next, the cells can be completely resuspended in a defined medium which enables the yeast cells to regenerate their cell wall. In order to select only those cells which have fused, a suitable selection pressure (for example a carbon source dependency for yeast and a low pH for <i>E. coli</i>) needs to be established.
              </p>
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          <ol class="literature_list">
-             <li id="ref_1"><a href="#ref_1" class="ref">[1]</a> RW, Brownsey, R. Zhande, and A. N. Boone. "lsoforms of acetyl-CoA carboxylase: structures, regulatory properties and metabolic functions." (1997).</li>
+             <li id="ref_1"><a href="#ref_1" class="ref">[1]</a> Agapakis, Christina M., et al. "Towards a synthetic chloroplast." PLoS One 6.4 (2011): e18877.</li>
-             <li id="ref_2"><a href="#ref_2" class="ref">[2]</a> Tehlivets, Oksana, Kim Scheuringer, and Sepp D. Kohlwein. "Fatty acid synthesis and elongation in yeast." Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1771.3 (2007): 255-270.</li>
+             <li id="ref_2"><a href="#ref_2" class="ref">[2]</a> Yamada, Takashi, and Kenji Sakaguchi. "Polyethylene glycol-induced uptake of bacteria into yeast protoplasts." Agricultural and Biological Chemistry 45.10 (1981): 2301-2309.</li>
-             <li id="ref_3"><a href="#ref_3" class="ref">[3]</a> Song, Chan Woo, et al. "Metabolic Engineering of Escherichia coli for the Production of 3-Hydroxypropionic Acid and Malonic Acid through β-Alanine Route." ACS synthetic biology (2016).</li>
+             <li id="ref_3"><a href="#ref_3" class="ref">[3]</a> Guerra-Tschuschke, I., I. Martin, and M. T. Gonzalez. "Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions." Applied and environmental microbiology 57.5 (1991): 1516-1522.</li>
-            <li id="ref_4"><a href="#ref_4" class="ref">[4]</a> Grobler, Jandre, et al. "The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids." Yeast 11.15 (1995): 1485-1491.</li>
+             <li id="ref_4"><a href="#ref_4" class="ref">[4]</a> Pastor, FI Javier, et al. "Structure of the Saccharomyces cerevisiae cell wall: mannoproteins released by zymolyase and their contribution to wall architecture." Biochimica et Biophysica Acta (BBA)-General Subjects 802.2 (1984): 292-300.</li>
-             <li id="ref_5"><a href="#ref_5" class="ref">[5]</a> Chen, Wei Ning, and Kee Yang Tan. "Malonate uptake and metabolism in Saccharomyces cerevisiae." Applied biochemistry and biotechnology 171.1 (2013): 44-62.</li>
+             <li id="ref_5"><a href="#ref_5" class="ref">[5]</a> Boni, Lawrence, et al. "Preparation of large liposomes by infusion into PEG." U.S. Patent Application No. 09/999,191.</li>
-            <li id="ref_6"><a href="#ref_6" class="ref">[6]</a> Kai, Yasushi, Hiroyoshi Matsumura, and Katsura Izui. "Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms."Archives of Biochemistry and Biophysics 414.2 (2003): 170-179.</li>
-             <li id="ref_7"><a href="#ref_7" class="ref">[7]</a> Song, Chan Woo, et al. "Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid." Metabolic engineering 30 (2015): 121-129.</li>
-            <li id="ref_8"><a href="#ref_8" class="ref">[8]</a> Zhang, Guijin, Stephen Brokx, and Joel H. Weiner. "Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli."Nature biotechnology 24.1 (2006): 100-104.</li>
-            <li id="ref_9"><a href="#ref_9" class="ref">[9]</a> Majander, Katariina, et al. "Extracellular secretion of polypeptides using a modified Escherichia coli flagellar secretion apparatus." Nature biotechnology23.4 (2005): 475-481.</li>
          </ol>

Difference between revisions of "Team:Marburg/PEG Method"

Revision as of 23:18, 19 October 2016

SynDustry Fuse. Use. Produce.

Fusion of different organisms using polyethylene glycol (PEG)

Literature