Team:TU Darmstadt/Project/Scaffold

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<!--TYPO3SEARCH_begin--><div id="c254" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Introduction</h1></div><p>It is important to address the concerns and possible consequences of intentional or accidental release into the environment when working with genetically engineered systems. These issues were already under discussion in the early years of Genetic Engineering and the Asilomar Conference in 1975 came to the conclusion that containment should always be made an important consideration in the design of the experiment. Furthermore should the effectiveness of the containment-approach match the estimated risk as closely as possible [1]. These principles still hold up until today and their importance even increased as the technology in recent years has shifted from the use of „simple“ transgenic&nbsp; organisms to designed synthetically constructs.</p></div><div id="c256" class="csc-default"><p>The goal of our iGEM safety project 2014 is to produce anthocyans in E.coli under laboratory or industrial conditions. Therefore our bacteria are not intended to be released into the environment and our considerations about biosafety are focused on preventing proliferation in case of an accidental release. To do so we decided to follow an active strategy for containment by designing a suicide construct that should prevent the survival of our cells if they get released into the environment but still does not limit the bacteria growth under controlled laboratory conditions. The main item of our suicide construct is the hokD gene of <i>E. coli</i> wich transcribes for a small polypeptide that results in cell death by elimination of vital cell wall functions if over expressed. The hokD gene is located in the <i>E. coli</i> chromosome as part of the relB operon. It’s gene product shows 40% homology to the polypeptide encoded by the hok (host killing) gene and induces the same characteristic cell deaths when over expressed [2]. It has been shown that the hokD gene can be used to design efficient suicide functions to contain bacteria growth. The efficiency of the containment system hereby is limited only by the mutation rate of the designed construct and the reduced growth rate resulting from a low basal level of <i>hokD</i>-expression [3, 4]. To avoid unwanted cell death due to basal expression we want to design a suicide backbone in wich hokD is under control of a T3 promotor (<i>phi4.3</i>) but the expression of T3-polymerase itself is controlled by an AraC-repressed pBad-promotor (BBa_K808000). In the presence of glucose the AraC repressor tightly inhibits the expression of T3-Polymerase and expression of hokD is inhibited so that the bacteria can grow with normal growth rate under controlled conditions [5]. In case of a release into the environment and upon depletion of glucose T3-polymerase is produced and the basal expression level of the T3 promotor results in expression of hokD and inhibits the proliferation of the bacteria.</p></div><div id="c263" class="csc-default"><div class="csc-textpic csc-textpic-center csc-textpic-above csc-textpic-equalheight"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><div class="csc-textpic-center-outer"><div class="csc-textpic-center-inner"><figure class="csc-textpic-image csc-textpic-last"><a href="index.php?eID=tx_cms_showpic&amp;file=246&amp;md5=d9b3918498f7db5ec1aafd19de808606d7fc6952&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D" onclick="openPic('index.php?eID=tx_cms_showpic&amp;file=246&amp;md5=d9b3918498f7db5ec1aafd19de808606d7fc6952&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D','thePicture','width=438,height=600,status=0,menubar=0'); return false;" target="thePicture"><img src="fileadmin/_processed_/csm_SafetySchaubild_afa1fc3d19.png" width="510" height="700" alt=""></a><figcaption class="csc-textpic-caption">Fig. 1: Schematics of our envisioned killing switch.</figcaption></figure></div></div></div></div></div><div id="c257" class="csc-default"><p>To evaluate the basal and induced expression levels of our suicide backbone we will first engineered a test backbone (pSB1C3-YAT) in which hokD is replaced by eYFP (BBa_E0030) (Fig. 2). By measuring the fluorescence level under different glucose and arabinose concentrations we are able to determine whether our construct is regulated enough to prevent unwanted cell death and the efficiency of containment after induction.</p></div><div id="c376" class="csc-default"><div class="csc-textpic csc-textpic-center csc-textpic-above csc-textpic-equalheight"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><div class="csc-textpic-center-outer"><div class="csc-textpic-center-inner"><figure class="csc-textpic-image csc-textpic-last"><a href="index.php?eID=tx_cms_showpic&amp;file=364&amp;md5=d7da9f47c56f5682340a3d0a3acb6dcd31959263&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D" onclick="openPic('index.php?eID=tx_cms_showpic&amp;file=364&amp;md5=d7da9f47c56f5682340a3d0a3acb6dcd31959263&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D','thePicture','width=723,height=600,status=0,menubar=0'); return false;" target="thePicture"><img src="fileadmin/_processed_/csm_pSB1C3-Yat_Map_6a00e234d8.png" width="301" height="250" alt=""></a><figcaption class="csc-textpic-caption">Fig. 2: Map of the planned test-plasmid (pSB1C3-YAT)</figcaption></figure></div></div></div></div></div><div id="c261" class="csc-default"><div class="csc-header csc-header-n6"><h1>References</h1></div><p>1.&nbsp;&nbsp; &nbsp;Berg, P., et al., Summary statement of the Asilomar conference on recombinant DNA molecules. Proc Natl Acad Sci U S A, 1975. 72(6): p. 1981-4.<br />2.&nbsp;&nbsp; &nbsp;Gerdes, K., et al., Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. Embo j, 1986. 5(8): p. 2023-9.<br />3.&nbsp;&nbsp; &nbsp;Knudsen, S.M. and O.H. Karlstrom, Development of efficient suicide mechanisms for biological containment of bacteria. Appl Environ Microbiol, 1991. 57(1): p. 85-92.<br />4.&nbsp;&nbsp; &nbsp;Knudsen, S., et al., Development and testing of improved suicide functions for biological containment of bacteria. Appl Environ Microbiol, 1995. 61(3): p. 985-91.<br />5.&nbsp;&nbsp; &nbsp;Saida, F., et al., Expression of highly toxic genes in E. coli: special strategies and genetic tools. Curr Protein Pept Sci, 2006. 7(1): p. 47-56.</p></div><!--TYPO3SEARCH_end-->
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<!--TYPO3SEARCH_begin--><div id="c198" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">The Scaffold Idea</h1></div><div class="csc-textpic-text"><div style="text-align: justify; "><p><span style="line-height: 18px; ">The steric proximity of enzymes can cause higher yield and production rates of final goods in metabolic processes. Different catalysation cascades can be found in nature that show intermediates that are transported to their next destination through channels or enzymes that are located closely to each other (Huang et al. 2001). This leads to an increased local concentration of metabolites near the enzymes und is especially useful in case of unstable or toxic intermediates of a reaction. To make use of such possibilities in synthetic biology, the Keasling Lab developed a construction of an easily and universally applicable protein scaffold. Its potency was proven in two examples with an up to 77-fold increase in yield (Dueber et al. 2009). The scaffold consists of three protein binding domains that may be linked by any number and in any order (Fig. 1). The scaffold can interlink with enzymes through domain-specific tags that are added to the catalytic proteins. The scaffold offers regulatory options for the stoichiometry of enzymes in steric proximity and may thereby counteract bottlenecks and excessive production of intermediates.</span></p></div></div></div><div id="c199" class="csc-default"><div class="csc-textpic csc-textpic-intext-right"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><figure class="csc-textpic-image csc-textpic-last"><a href="index.php?eID=tx_cms_showpic&amp;file=162&amp;md5=22d466c6236c31dffad8191b710cb23ebe8cfe96&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D" onclick="openPic('index.php?eID=tx_cms_showpic&amp;file=162&amp;md5=22d466c6236c31dffad8191b710cb23ebe8cfe96&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D','thePicture','width=800,height=422,status=0,menubar=0'); return false;" target="thePicture"><img src="fileadmin/_processed_/csm_Scaffold_skizze__Fig1__bearbeitet12_bc598ccc94.png" width="300" height="159" alt=""></a></figure></div><div class="csc-textpic-text"><p>Fig 1: The scaffold molecule consists of three different interaction-domains, that can be freely combined (x, y, z). Chosen enzymes get attached to the scaffold through a tag complementary to the specific desired binding domains. This arrangement creates a production chain which generates higher yields.</p></div></div></div><div id="c201" class="csc-default"><div class="csc-textpic-text"><p>As far as we know, there have not been any successful attempts to clarify the structure of the scaffold molecule. NMR-structures of the separate parts (binding domains: GBD, PDZ, SH3) are known and were used to predict a homology-modeling based structure using PHYRE2 (Fig. 2).</p></div></div><div id="c202" class="csc-default"><div class="csc-textpic csc-textpic-intext-right"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><figure class="csc-textpic-image csc-textpic-last"><a href="index.php?eID=tx_cms_showpic&amp;file=164&amp;md5=0bffed4b97800ec40ba6a99350b40a26b7c2df03&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D" onclick="openPic('index.php?eID=tx_cms_showpic&amp;file=164&amp;md5=0bffed4b97800ec40ba6a99350b40a26b7c2df03&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D','thePicture','width=800,height=468,status=0,menubar=0'); return false;" target="thePicture"><img src="fileadmin/_processed_/csm_Scaffold__Fig2__barbeitet12_b41ccbf035.png" width="300" height="176" alt=""></a></figure></div><div class="csc-textpic-text"><p>Fig. 2: 3D-structure of the protein scaffold, created with PHYRE2 based on structure-homology-modeling.</p></div></div></div><div id="c203" class="csc-default"><div class="csc-textpic-text"><div><p>The use of a scaffold protein might lead to a significant increase of yield of the anthocyanidin that we want to produce in <i>E. coli</i> in our 2014 iGEM project. Since especially DFR of the enzymatic cascade shows only a relatively small turnover rate and also catalyzes several side reactions, it might be the limiting step for the overall production rate of our pathway. For this reason, we tried to establish a method to produce and characterize the scaffold for a further usage as a regulatory device and an improvement of our metabolic network. &nbsp;</p></div><div></div><div><p>We tried to establish a system for the covalent crosslinking of our scaffold using the thiole group of a cysteine. Cysteine is not present in the sequence of the scaffold protein and therefore enables regiospecific coupling reactions. Cysteines belong to the naturally occurring amino acids and are of great significance for protein folding. Two cysteins can form a disulfide bond that leads to structural constraints while the folding-process of a protein takes place. Cysteines are a popular target for chemical modifications of proteins and bioconjugation due to their unique reactivity among amino acids. Generally, cysteine is one of the most used amino acid residue for protein modifications. Proteins can be provided with a virtually unlimited variety of modifications using coupling reagents as maleimids, haloacetamids etc. A frequent utilization of such free cysteine residues is protein immobilization on resin particles or the labeling of proteins with fluorophors. Bioconjugation of proteins with fluorophors is of great use for binding studies. Mainly, proteins with only one accessible cysteine residue are of interest for coupling reactions since a regioselective reaction is not possible if multiple cystein residues are present in a reactive and accessible form.</p></div><div></div></div></div><div id="c204" class="csc-default"><div class="csc-textpic csc-textpic-left csc-textpic-above"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><figure class="csc-textpic-image csc-textpic-last"><a href="index.php?eID=tx_cms_showpic&amp;file=183&amp;md5=a0d6d69e463ef82074d57b34e60a34d42760f690&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D" onclick="openPic('index.php?eID=tx_cms_showpic&amp;file=183&amp;md5=a0d6d69e463ef82074d57b34e60a34d42760f690&amp;parameters%5B0%5D=YTo0OntzOjU6IndpZHRoIjtzOjQ6IjgwMG0iO3M6NjoiaGVpZ2h0IjtzOjQ6IjYw&amp;parameters%5B1%5D=MG0iO3M6NzoiYm9keVRhZyI7czo0MToiPGJvZHkgc3R5bGU9Im1hcmdpbjowOyBi&amp;parameters%5B2%5D=YWNrZ3JvdW5kOiNmZmY7Ij4iO3M6NDoid3JhcCI7czozNzoiPGEgaHJlZj0iamF2&amp;parameters%5B3%5D=YXNjcmlwdDpjbG9zZSgpOyI%2BIHwgPC9hPiI7fQ%3D%3D','thePicture','width=800,height=164,status=0,menubar=0'); return false;" target="thePicture"><img src="fileadmin/_processed_/csm_Scaffold_Fig3__english__046904995c.png" width="600" height="123" alt=""></a></figure></div><div class="csc-textpic-text"><p style="text-align:justify"><span lang="EN-GB">Fig. 3: Mechanism of an immobilization reaction. A free cysteine residue of a protein reacts with the maleimide function on the surface of a resin particle.</span></p></div></div></div><div id="c216" class="csc-default"><div class="csc-header csc-header-n7"><h1>References</h1></div><div class="csc-textpic-text"><div><p>Xinyi Huang, Hazel M. Holden, and Frank M. Raushel, &quot;Channeling of substrates und intermediates in enzyme-catalyzed reactions&quot;. Annual review of biochemistry, 70: 149 - 180, 2001.</p></div><div></div><div><p>John E. Dueber, Gabriel C. Wu, G. Reza Malmirchegini, Tae Seok Moon, Christopher J. Petzold, Adeeti V. Ullal, Kristala L.J. Prather, und Jay D. Keasling, &quot;Synthetic protein scaffolds provide modular control over metabolic flux&quot;. Nature biotechnology, 27: 753 - 759, 2009.</p></div></div></div><!--TYPO3SEARCH_end-->
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The Scaffold Idea

The steric proximity of enzymes can cause higher yield and production rates of final goods in metabolic processes. Different catalysation cascades can be found in nature that show intermediates that are transported to their next destination through channels or enzymes that are located closely to each other (Huang et al. 2001). This leads to an increased local concentration of metabolites near the enzymes und is especially useful in case of unstable or toxic intermediates of a reaction. To make use of such possibilities in synthetic biology, the Keasling Lab developed a construction of an easily and universally applicable protein scaffold. Its potency was proven in two examples with an up to 77-fold increase in yield (Dueber et al. 2009). The scaffold consists of three protein binding domains that may be linked by any number and in any order (Fig. 1). The scaffold can interlink with enzymes through domain-specific tags that are added to the catalytic proteins. The scaffold offers regulatory options for the stoichiometry of enzymes in steric proximity and may thereby counteract bottlenecks and excessive production of intermediates.

Fig 1: The scaffold molecule consists of three different interaction-domains, that can be freely combined (x, y, z). Chosen enzymes get attached to the scaffold through a tag complementary to the specific desired binding domains. This arrangement creates a production chain which generates higher yields.

As far as we know, there have not been any successful attempts to clarify the structure of the scaffold molecule. NMR-structures of the separate parts (binding domains: GBD, PDZ, SH3) are known and were used to predict a homology-modeling based structure using PHYRE2 (Fig. 2).

Fig. 2: 3D-structure of the protein scaffold, created with PHYRE2 based on structure-homology-modeling.

The use of a scaffold protein might lead to a significant increase of yield of the anthocyanidin that we want to produce in E. coli in our 2014 iGEM project. Since especially DFR of the enzymatic cascade shows only a relatively small turnover rate and also catalyzes several side reactions, it might be the limiting step for the overall production rate of our pathway. For this reason, we tried to establish a method to produce and characterize the scaffold for a further usage as a regulatory device and an improvement of our metabolic network.  

We tried to establish a system for the covalent crosslinking of our scaffold using the thiole group of a cysteine. Cysteine is not present in the sequence of the scaffold protein and therefore enables regiospecific coupling reactions. Cysteines belong to the naturally occurring amino acids and are of great significance for protein folding. Two cysteins can form a disulfide bond that leads to structural constraints while the folding-process of a protein takes place. Cysteines are a popular target for chemical modifications of proteins and bioconjugation due to their unique reactivity among amino acids. Generally, cysteine is one of the most used amino acid residue for protein modifications. Proteins can be provided with a virtually unlimited variety of modifications using coupling reagents as maleimids, haloacetamids etc. A frequent utilization of such free cysteine residues is protein immobilization on resin particles or the labeling of proteins with fluorophors. Bioconjugation of proteins with fluorophors is of great use for binding studies. Mainly, proteins with only one accessible cysteine residue are of interest for coupling reactions since a regioselective reaction is not possible if multiple cystein residues are present in a reactive and accessible form.

Fig. 3: Mechanism of an immobilization reaction. A free cysteine residue of a protein reacts with the maleimide function on the surface of a resin particle.

References

Xinyi Huang, Hazel M. Holden, and Frank M. Raushel, "Channeling of substrates und intermediates in enzyme-catalyzed reactions". Annual review of biochemistry, 70: 149 - 180, 2001.

John E. Dueber, Gabriel C. Wu, G. Reza Malmirchegini, Tae Seok Moon, Christopher J. Petzold, Adeeti V. Ullal, Kristala L.J. Prather, und Jay D. Keasling, "Synthetic protein scaffolds provide modular control over metabolic flux". Nature biotechnology, 27: 753 - 759, 2009.