Team:Freiburg bioware/Project

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Expression and Purification</a></li>
Expression and Purification</a></li>
           <li><a href="#In_vivo_Expression">In vivo
           <li><a href="#In_vivo_Expression">In vivo
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Expression</a></li>
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assays</a></li>
           <li><a href="#AGO">AGO</a></li>
           <li><a href="#AGO">AGO</a></li>
           <li><a href="#FOS">FOS</a></li>
           <li><a href="#FOS">FOS</a></li>
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<a name="Summary"></a>Summary<span
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We focused our project on coupling and optimizing the characteristics
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of a restriction endonuclease with short oligonucleotides to develop a
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The goal of providing an universal restriction enzyme was approached
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programmable and highly specific enzyme-oligo-complex. As a restriction
+
with two design strategies. The first strategy evolves around novel
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endonuclease we chose the cleavage domain of the well studied
+
protein fusion constructs combining the cleavage domain of the Type IIs
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endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts
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restriction enzyme FokI with hapten binding anticalins, which are then
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as a homodimer, each dimer divided in cleavage and restriction domain.
+
guided to their target sites by a modified oligonucleotide. The second
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Chandrasegaran and Miller have already made experiments to uncouple the
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approach aimed at converting the Argonaute proteins from thermophilic
 +
organisms from an RNase to an DNase activity while accepting a DNA
 +
guide oligonucleotide. For the Fok-based strategy, several variations
 +
were tested. In both projects important milestones were reached. Most
 +
importantly, we demonstrated guided cleavage by one of our Fok-based
 +
fusion constructs and phage display of an Argonaute protein.
 +
In the following we introduce these two projects and
 +
then list the highlights of our work with links to detailed
 +
descriptions of each project part. <br />
 +
Both strategies rely on locating the universal restriction enzyme at
 +
the cleavage site with adapter or guide oligonucleotides. This is in
 +
contrast to previous designs which either use chemical linkage of an
 +
oligonucleotide to a nuclease or genetic fusion with a DNA binding
 +
domain. Previous fusion protein approaches have the conceptual
 +
disadvantage that for each target sequence a special protein has to be
 +
designed, expressed, purified, and stored. We only need to produce one
 +
protein. The oligonucleotide planning is aided by readily available PCR
 +
primer design programs and there are virtually no limits to define the
 +
cleavage site. Modified oligonucleotides can be ordered from many
 +
suppliers at low cost with short delivery times and easily can be
 +
stored long term. In in-vitro applications hybridization of the
 +
oligonucleotide is easily achieved also to double strand by heating and
 +
cooling as it is well known from PCR procedures. In case of the
 +
Argonaute proteins from thermophiles we can assume that they survive
 +
several cycles of heat dissociation and annealing. In case of the Fok
 +
we plan to introduce thermostability by directed evolution. In
 +
addition, other groups are working with oligonucleotides forming triple
 +
helices and their general hybridization with any sequence or with
 +
peptide nucleotide acids which provide higher stability and sneak in
 +
existing double helices. These technologies are compatible with our
 +
approach.<br />
 +
In both of our universal restriction enzyme strategies we do not cut
 +
the double strand but rather nick the stand opposite to our guide
 +
oligonucleotide. Thus, our guide oligonucleotide only needs to be added
 +
only in catalytic quantities. The nicking feature is already present in
 +
the Argonaute proteins. In the case of the universal Fok-based enzyme
 +
we use a heterodimer design combined with cleavage inactivating
 +
mutations for one monomer.
 +
<br /><br />
 +
<strong>Details of the universal restriction enzyme based on Fok-Anticalin fusions</strong><br />
 +
The restriction endonuclease FokI from Flavobacterium okeanokoites is a
 +
well studied protein. It consists of two domains, a DNA recognition
 +
domain and a DNA cleavage domain. Upon recognition of one target site
 +
and dimerization it cleaves the DNA nine bases apart from the
 +
recognition site. Several groups reported experiments to uncouple the
cleavage and restriction domains of FokI and created a novel
cleavage and restriction domains of FokI and created a novel
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site-specific endonuclease by linking the cleavage domain to zinc
+
site-specific endonucleases by linking the cleavage domain to zinc
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finger proteins.<br />
+
finger proteins (Miller et al. 2007). <br />
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For our project we generated two Fok heterodimers (Miller, Nature
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For our project we combined two previous research results and generated
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biotech, 2007). For the catalytic active Fok partner, named Fok_a, the
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a Fok cleavage heterodimer comprising an enzymatically active and
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first 1158 nucleotides, i.e. the recognition domain, were deleted and
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inactive monomer. For the catalytic active Fok partner, named Fok_a, as
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glutamate 490 was switched to lysine (GAA-&gt;AAA) as well as
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well as for the catalytic inactive Fok partner, Fok_i, the association
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isoleucine 538 to lysine (ATC-&gt;AAA) for the heterodimer
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interface was mutated to disfavor homodimerization and promote
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formation.
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heterodimerization. In Fok_i additional amino acid exchanges led to the
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For the catalytic inactive Fok partner, named Fok_i, the heterodimeric
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inactivation.<br />
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amino acids glutamine 486 was switched to glutamate (CAA-&gt;GAA)
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The two heterodimeric partners were fused to anticalins binding
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and
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different adapter molecules. Fok_a is genetically fused to a
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isoleucine 499 to leucine (ATC-&gt;CTG) and the catalytic amino
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digoxigenin-binding anticalin (DigA) and Fok_i to a fluorescein-binding
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acids
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anticalin (FluA). The adapter molecules digoxigenin and fluorescein are
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aspartate 450 was switched to alanine (GAC-&gt;GCG) and aspartate
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common modifications linked to oligonucleotides thus mediating the
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467
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binding to the DNA site of interest. Two modifications allow for a
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to alanine (GAT-&gt;GCG).<br />
+
better spatial control of the cleavage site. On the target site Fok_i
 +
and Fok_a constructs are brought into close proximity and can form a
 +
heterodimer. The inactive Fok domain will serve as an activator of the
 +
active Fok domain, which cuts one strand of the DNA. Our structural
 +
&lsquo;3D&rsquo; models, indicate that Fok domains can be
 +
positioned in such a way that Fok_a will cleave the target DNA, and
 +
Fok_i would be directed towards the modified oligonucleotide. Different
 +
linkers were designed and fused between cleavage domain and the
 +
anticalin binding moieties to test for the optimal distance. In
 +
addition we generated a monomeric Fok fusion construct, to enable phage
 +
display and te4st a further setting requiring only one binding domain
 +
and hapten. Furthermore, we made a Fok cleavage domain fusion with a
 +
coiled coil based DNA binding domain, because we can combine these with
 +
existing light switchable inhibitors, which prevent DNA binding. As a
 +
result we will obtaina light switchable restriction enzyme.
 +
<br /><br />
 +
<strong>Milestones</strong> <br />
 +
To reach our goal within the short given time frame we started several
 +
subprojects in parallel. Our subprojects listed here are defined along
 +
these projects. <br />
 +
Designing of the constructs was aided by extensive model building and
 +
analysis of the spatial orientation of the different proteins and
 +
oligonucleotides used. All designed and constructed parts feature full
 +
BioBrick compatibility and in addition allow for the construction of
 +
fusion proteins based on the RFC 25 (Freiburg) cloning standard.
 +
Cloning of the respective parts was followed by expression purification
 +
and analysis. Despite previous literature data our active Fok construct
 +
was toxic to cells when expressed and we tested
 +
periplasmic expression with export of the nascent polypeptide
 +
chain before folding.<br />
 +
 
 +
In our experiments we addressed the following questions:<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Structural Model building<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Design of protein fusion parts<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Cloning of anticalin Fok fusions<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Cloning of a monomeric Fok construct and of a Jun/Fos directed Fok construct<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Expression and purification of constructs<br />
 +
&bull;&nbsp;&nbsp;&nbsp; <em>In vitro</em> assays<br />
 +
&bull;&nbsp;&nbsp;&nbsp; <em>In vivo</em> assays<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Phage Display of an Ago protein<br />
 +
&bull;&nbsp;&nbsp;&nbsp; Modeling of assembly and cleavage with differential equations<br />
 +
&bull;&nbsp;&nbsp;&nbsp; An international survey of laymen on synthetic biology<br />
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In short, we successfully worked on all aspects. Experiments to validate our approach in more compelx settings are ongoing. <br />
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The labs of Kristian M&uuml;ller and Katja Arndt provided all technology and support for the project.<br />
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      <td><small>Association of linker FluA and Dig with
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DNA and</small> <small>Fok_a
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and Fok_i monomers</small></td>
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<div style="text-align: justify;">The two heterodimeric
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partners were fused to different anticalins
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binding different adapter molecules. Thus Fok_i is fused to anticalin
+
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on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter
+
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molecules are linked to oligonucleotides mediating the binding of the
+
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DNA site of interest. Now the heterodimerization comes into play. If
+
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the different Fok_i and Fok_a constructs bind their target oligos and
+
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come together, the inactive domain will serve simply as an activator of
+
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the active domain, cutting only one strand of the DNA. In our 3D models
+
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we showed that Fok domains are positioned in such a way that Fok_a will
+
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cut the DNA and Fok_i the modified oligonucleotide. Thus the
+
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inactivation of Fok_i allows the reuse of our oligonucleotides.
+
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Different linkers were designed and fused between cleavage domain and
+
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binding protein to test the optimal distance to preserve the most
+
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possible flexibility and most possible precision of the heterodimeric
+
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Foks.<br />
+
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       <td><img style="width: 356px; height: 240px;" alt=""
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src="https://static.igem.org/mediawiki/2009/5/5e/Freiburg09_fokmodel_fig4.png" /><br />
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      src="https://static.igem.org/mediawiki/2009/2/2b/Freiburg_09_Foki_foka_schema.JPG" /><br />
 +
      Schematic Model of the universal restriction enzymes based on<br />
 +
      FokI and anticalins.
 +
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 +
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 +
      <img alt="" src="https://static.igem.org/mediawiki/2009/7/71/Freiburg09_UniFok_model.png" /><br />
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      Structural model of the universal restriction enzymes based on FokI and anticalins.
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       <td><small>complete universal restriction enzyme.
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       <td><img style="width: 357px; height: 249px;" alt=""
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Blue: DNA strand; Red: two 16bp
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      src="https://static.igem.org/mediawiki/2009/d/d7/Freiburg09_Scheme_binding_cutting_melting.JPG" /><br />
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long Oligos,</small> <small>tags as indicated in the
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      Model of the catalytic cycle; hybridization - cleavage - temperature<br />
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picture. Ochre: Fluorescein A
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      promoted release.
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binding lipocalin; Orange: digoxigenin</small> <small>binding
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lipocalin; Yellow:
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tagged Base with C6 linkers and attached tags; light Blue: inactive
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      src="https://static.igem.org/mediawiki/2009/5/54/Freiburg09_Batz.png" /><br />
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FokI</small> <small>cleavage domain; Red: FokI active
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      Structure of an Ago protein, demonstrating guide oligonucleotide<br />
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cleavage domain; Green: three
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      mediated binding.
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catalytically active aminoacids; White: two Calcium ions</small></td>
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<div style="text-align: justify;">The place where all the
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cutting events will take place in our scenery
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is the thermocycler. Therefore all the ingredients, i.e. chromosomal or
+
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plasmid DNA of interest, modified oligonucleotides and the different
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heterodimers Fok_a and Fok_i are mixed together. At high temperatures
+
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the DNA will denaturate allowing the different partners to find each
+
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other, cut the DNA, and fall apart in course of one thermocycle. The
+
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whole procedure can be repeated undefinately. To reach this step, we
+
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need to improve the thermostability of our enzyme via phage display.
+
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Creating a universal restriction enzyme provides not only the
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possibility to improve routine cloning but also to enhance therapeutic
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gene repair via triplex technology. Many genetic diseases and
+
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especially ones arising from single nucleotide polymorphisms (SNPs) or
+
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monogenetic disease can be alleviated by the replacement of mutated
+
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genes using this method. To cut double stranded DNA the
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oligonucleotides have to be replaced by triple helix forming oligos
+
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(TFO). They can bind double-stranded DNA in homopurin- or
+
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homopyrimidine-rich areas. But developments are also made to widen the
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possible interaction domains of the DNA and hence make the TFOs as
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programmable as our conventional oligonucleotides. In case of the human
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genome of 3&times;10^9 bp size, a highly specific artifical
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endonuclease
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would be necessary to address the mutated gene explicitly. The used
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TFOs therefore have to possess a minimum length of 16 bp to cut just
+
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once in the human genome (4^16 bp = 4.3*10^9 bp).<br />
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<div style="text-align: justify;">Additionally, we provide
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an alternative way of binding by the coupling
+
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of the cleavage domain to the transcription factor Fos. This concept
+
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also includes the activity regulation by photo-switching. We are also
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modifying an argonaute protein into a DNA endonuclease using phage
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display technics.
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<h4><a name="Modelling"></a>Modeling</h4>
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<a href="#">Read more...</a>
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<h4><a name="Modelling"></a>Modeling of the Enzyme Kinetics</h4>
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<h4><a name="3D-Modelling"></a>3D-Modeling</h4>
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      <td style="text-align:justify; padding: 0.5em;">Structural modeling was an initial step towards our planned universal restriction enzyme. Using molecular display software we arranged published crystal structures of FokI, anticalins and DNA. The arrangement was guided by superimposition with further structures of enzyme bound DNA. The modeling defined spatial requirements for linker lengths and positioning of modifications within oligonucleotides.</td>
+
    <td>For our modeling analyses we constructed various sets of differential equations describing
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      <td style="text-align: right;"><img
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    protein-protein and protein-DNA interactions and the final cleavage. <br />
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style="width: 300px; height: 203px;" alt=""
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<a href="https://2009.igem.org/Team:Freiburg_bioware/Modeling">Read more...</a>
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src="https://static.igem.org/mediawiki/2009/thumb/1/11/Freiburg09_fokmodel_completeFok.png/800px-Freiburg09_fokmodel_completeFok.png" /></td>
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<a
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href="https://2009.igem.org/Team:Freiburg_bioware/Project/3d-modeling">Read
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<hr style="width: 100%; height: 2px;" />
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<h4><a name="Fok_a"></a>In vitro assays</h4>
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<h4>Modeling of the Enzyme Structure</h4>
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      Structural modeling was an initial step towards
 +
      our planned universal restriction enzyme. Using molecular display software we arranged published
 +
      crystal structures of FokI, anticalins and DNA. The arrangement was guided by superimposition with
 +
      further structures of enzyme bound DNA. The modeling defined spatial requirements for linker lengths
 +
      and positioning of modifications within oligonucleotides.
 +
      <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/3d-modeling">Read more...</a></td>
 +
      <td style="text-align: right;"><img style="width: 300px; height: 203px;" alt=""
 +
      src="https://static.igem.org/mediawiki/2009/thumb/1/11/Freiburg09_fokmodel_completeFok.png/800px-Freiburg09_fokmodel_completeFok.png" /></td>
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<hr style="width: 100%; height: 2px;" />
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<h4><a name="Fok_a"></a><i>In vitro</i> assays</h4>
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       <td>After the cloning, expression and the purification of
+
       <td>
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the Fok
+
      After the cloning, expression and the purification of
-
constructs we conducted several assays to analyse the activity of the
+
      the Fok constructs we conducted several assays in order to to analyze the activity of the enzyme. To
-
enzyme. To establish the assay and as a reference for activity we used
+
      establish the
-
wildtype FokI. Binding of the modified nucleotides and enzymatic
+
      assay and as a reference for activity we used wild type FokI. Binding of the modified  
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activity were tested with the Fok_i / Fok_a construct.
+
      nucleotides and enzymatic activity were tested with the Fok_i / Fok_a construct.
 +
      <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/invitro">Read more...</a>
       </td>
       </td>
-
       <td style="text-align: right;"><img
+
       <td style="text-align: right;"><img  style="width: 75px; height: 185px;" alt=""
-
  style="width: 271px; height: 148px;" alt=""
+
      src="https://static.igem.org/mediawiki/2009/0/00/Freiburg09_Fok_Winner_FluoMikroskop_only.jpg" /></td>
-
src="https://static.igem.org/mediawiki/2009/9/9e/Freiburg09_Invitro_cropped.jpg" /></td>
+
      <td style="text-align: right;"><img style="width: 75px; height: 185px;" alt=""
 +
      src="https://static.igem.org/mediawiki/2009/1/18/Freiburg09_Fok_80mer_in_vitro_cutting_assay_EtBr.jpg" /></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a href="https://2009.igem.org/Team:Freiburg_bioware/Project/invitro">Read
+
 
-
more...</a>
+
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
-
<h4><a name="Fok_Monomer"></a>Fok Monomer</h4>
+
 
-
<br />
+
<h4><a name="In_vivo_Expression"></a><i>In vivo</i> Assays</h4>
-
<table style="text-align: left; width: 900px;" border="0"
+
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
-
cellpadding="0" cellspacing="0">
+
 
-
  <tbody bgcolor="#e2eff9">
+
     <tr>
     <tr>
-
       <td><img alt=""
+
       <td><a href=""><img style="border: 0px solid ; width: 175px; height: 125px;" alt=""  
-
src="/wiki/index.php?title=User:Maximi/new_general_style.css&amp;action=raw&amp;ctype=text/css" /><img
+
      src="https://static.igem.org/mediawiki/2009/9/9a/Freiburg_09_2009-09-21_multidimensional_RV308_electro_mit_fluo_oligos-4_c1.JPG" /></a>
-
style="width: 300px; height: 150px;" alt=""
+
      </td>
-
src="https://static.igem.org/mediawiki/2009/2/29/Freiburg09_Monomermodel1.JPG" /></td>
+
       <td>
       <td>
-
       <p class="MsoNormal" style="text-align: justify;"><span
+
       We demonstrated that modified guide oligonucleotides can be transfected in E. coli.
-
style="" lang="EN-GB">In order to create a universal
+
      Importantly, we showed that a M13 DNA hybridized with a guide oligonucleotide does
-
restriction
+
      not produce phages when transfectd in cells expressing Fok fusion proteins whereas
-
enzyme we followed several ideas and models. The first and most
+
      the control M13 DNA does.
-
promising idea
+
        
-
we had was to create a Fok Monomer as it represents the optimal and
+
      <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/invivo">Read more...</a>
-
easiest
+
-
model for a universal restriction enzyme<o:p></o:p><u><span
+
-
style="" lang="EN-GB"><o:p><span
+
-
style="text-decoration: none;"></span></o:p></span></u></p>
+
-
      <p class="MsoNormal" style="text-align: justify;"><u><span
+
-
style="" lang="EN-GB"><o:p><span
+
-
style="text-decoration: none;"></span></o:p></span></u><span
+
-
style="" lang="EN-GB">If we are able to employ a
+
-
monomeric enzyme,
+
-
this protein would have a couple of advantages. Most importantly we
+
-
would no longer
+
-
need two separate oligonucleotides to achieve specific binding and
+
-
cutting of
+
-
the target DNA. Also only one protein has to be purified &ndash;
+
-
thus saving time and
+
-
money. A scientific advantage is the option to optimize the monomer by
+
-
phage
+
-
display. Using this technology, we would have the chance to create a
+
-
thermostable, specific, universal restriction enzyme whose DNA binding
+
-
activity
+
-
is only created by a single oligonucleotide.<o:p></o:p></span><u><span
+
-
style="" lang="EN-GB"><o:p><span
+
-
style="text-decoration: none;"></span></o:p></span></u></p>
+
-
       <p class="MsoNormal" style="text-align: justify;"><u><span
+
-
style="" lang="EN-GB"><o:p><span
+
-
style="text-decoration: none;"></span></o:p></span></u><span
+
-
style="" lang="EN-GB">Our goal was firstly to create
+
-
a Fok-monomer which
+
-
is able to cut DNA without a primary dimerization step and secondly to
+
-
show
+
-
that our heterodimeric interface design works properly. To reach this,
+
-
we had
+
-
to clone all the required parts one after another, resulting in a huge
+
-
fusion
+
-
protein.<o:p></o:p></span></p>
+
       </td>
       </td>
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a
+
 
-
href="https://2009.igem.org/Team:Freiburg_bioware/Project/fokmonomer">Read
+
-
more...</a>
+
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
-
<h4><a name="Purification"></a>Protein
+
 
-
Expression and Purification</h4>
+
 
-
<br />
+
<h4><a name="Purification"></a>Protein Expression and Purification</h4>
-
<table style="text-align: left; width: 900px;" border="0"
+
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
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cellpadding="0" cellspacing="0">
+
 
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  <tbody bgcolor="#e2eff9">
+
     <tr>
     <tr>
       <td>
       <td>
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       <p class="MsoNormal" style="text-align: justify;"><span style=""
+
       Proteins were recombinantly expressed in E. coli strains BL21 or RV308. Purification was achieved by
-
lang="EN-US">In order to
+
      affinity chromatography utilizing the His-tag, Strep-tag, or the GST fusion. If needed, two affinity
-
produce and study our different protein constructs they had to be
+
      purifications due to two available tags were combined or a size exclusion chromatogrpahy was added.
-
expressed in
+
      <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/purification">Read more...</a>
-
bacteria.<span style="">&nbsp; </span>After
+
      </td>
-
cloning the individual
+
      <td><img style="width: 163px; height: 122px;" alt="" src="https://static.igem.org/mediawiki/2009/8/81/Freiburg_09_2009-10-19_BL21_FokA-YFP_induziert_(c4%2Bc5).JPG" />
-
parts into the pMA vector the complete expression products were then
+
-
transferred
+
-
into the pEx vector (see pEx vector ) and transformed into competent <i
+
-
style="">E. coli</i> expression strains via heat
+
-
shock. We used two different strains for the protein expression: <i
+
-
style="">E. coli</i> BL21 de3 (Novagen) and <i
+
-
style="">E. coli</i> RV308 (Maurer <i style="">et
+
-
al.</i>, JMB 1980). Both strains are suited
+
-
for high-level protein expression.<o:p></o:p></span></p>
+
       </td>
       </td>
-
      <td><img style="width: 163px; height: 122px;"
 
-
alt=""
 
-
src="https://static.igem.org/mediawiki/2009/a/a6/Freiburg09_RV308_mit_fluo_Oligos3_%28c1%2Bc2%29.JPG" /></td>
 
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a
+
 
-
href="https://2009.igem.org/Team:Freiburg_bioware/Project/purification">Read
+
-
more...</a>
+
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
-
<h4><a name="In_vivo_Expression"></a>In vivo
+
 
-
Expression</h4>
+
 
-
<br />
+
<h4><a name="AGO"></a>AGO</h4>
-
<table style="text-align: left; width: 900px;" border="0"
+
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
-
cellpadding="0" cellspacing="0">
+
 
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  <tbody bgcolor="#e2eff9">
+
     <tr>
     <tr>
-
       <td><a
+
       <td>
-
href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"><img
+
      The Argonaute proteins represent our second approach to generate universal restriction enzymes.
-
style="border: 0px solid ; width: 218px; height: 117px;" alt=""
+
      These RNases provide already important features such as recognition of guide oligonucleotides  and thermostability.
-
src="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG" /></a><a
+
      We expressed and purified an AGO protein and showed residual DNase activtiy. Consequently, we started
-
href="https://static.igem.org/mediawiki/2009/1/1c/Freiburg09_17-10-09_Phagenplatten_XGal.JPG"
+
      phage display of error prone PCR diversified AGO in order to convert the RNase in a DNase. We
-
target="_blank"><br />
+
      demonstratd AGO display, successfully completed two rounds of phage display and identified interesting mutants.
-
      </a></td>
+
      <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/AGO">Read more...</a>
-
      <td><span
+
      </td>
-
style=" line-height: 115%; ">Additionally
+
      <td><img style="width: 292px; height: 171px;" alt=""
-
to the in vitro assays with the directly purified programmable
+
      src="https://static.igem.org/mediawiki/2009/7/72/Freiburg09_AGOttstucturesceme.png" /></td>
-
restriction
+
-
endonuclease and isolated target <span style="">&nbsp;</span>DNA,
+
-
we
+
-
developed an in vivo assay. This means the experiments with Fok
+
-
constructs and
+
-
DNA in living <i style="">E. colis</i>. </span></td>
+
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a href="https://2009.igem.org/Team:Freiburg_bioware/Project/invivo">Read
+
 
-
more...</a>
+
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
-
<h4><a name="AGO"></a>AGO</h4>
+
 
-
<table style="text-align: left; width: 900px;" border="0"
+
 
-
cellpadding="0" cellspacing="0">
+
<h4><a name="Ethics"></a>Ethics</h4>
-
   <tbody bgcolor="#e2eff9">
+
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
 +
 
 +
    <tr>
 +
    <td>
 +
    We conducted an international survey on benefit and risk perception of Synthetic Biology by laymen, which was translated in 10 languages.
 +
    <a href="https://2009.igem.org/Team:Freiburg_bioware/Human_Practice/Ethics">Read more...</a>
 +
    </td>
 +
   </tr>
 +
  </tbody>
 +
</table>
 +
 
 +
<hr style="width: 100%; height: 2px;" />
 +
 
 +
<h4><a name="Fok_Monomer"></a>Fok Monomer</h4>
 +
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
     <tr>
     <tr>
       <td>
       <td>
-
       <p class="MsoNormal" style="text-align: justify;"><span style=""
+
       <img style="width: 300px; height: 150px;" alt=""  src="https://static.igem.org/mediawiki/2009/2/29/Freiburg09_Monomermodel1.JPG" /></td>
-
  lang="EN-US">The
+
      <td>
-
argonaute proteins represent one of our side projects in creating a
+
      To ease production, purification, handling and analysis we cloned a monomeric Fok variant.
-
universal
+
      In addition, this variant is better suited for phage display, which will allow us selection for
-
programmable endonuclease.<o:p></o:p></span></p>
+
      improved properties. This protein comprises four functional subunits interspaced by two short and
 +
      one long linker (anticalin-Fok_i-long_linker-Fok_a-anticalin).
 +
      <a  href="https://2009.igem.org/Team:Freiburg_bioware/Project/fokmonomer">Read more...</a>
       </td>
       </td>
-
      <td><img style="width: 292px; height: 171px;"
 
-
alt=""
 
-
src="https://static.igem.org/mediawiki/2009/7/72/Freiburg09_AGOttstucturesceme.png" /></td>
 
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a href="https://2009.igem.org/Team:Freiburg_bioware/Project/AGO">Read
+
 
-
more...</a>
+
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
-
<h4><a name="FOS"></a>Alternative way
+
 
-
of binding: Jun/Fos</h4>
+
<h4><a name="FOS"></a>Alternative way of binding: Jun/Fos</h4>
-
<table style="text-align: left; width: 910px; height: 138px;"
+
<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
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border="0" cellpadding="0" cellspacing="0">
+
       <td><img style="width: 200px; height: 200px;" alt=""
-
  <tbody bgcolor="#e2eff9">
+
      src="https://static.igem.org/mediawiki/2009/e/e1/Freiburg09_Fos.fosw.scheme.png" /></td>
-
    <tr>
+
       <td> As an alternative way of binding of Fok to DNA we cloned the DNA  
-
       <td><img style="width: 140px; height: 140px;"
+
      binding domain (bZIP) of the activator protein-1 (AP-1). As light switching of coiled coils
-
alt=""
+
      is established in our host lab, this will give us a light switchable restriction enzyme.  
-
src="https://static.igem.org/mediawiki/2009/5/57/Freiburg09_1FOS.jpg" /></td>
+
    <a href="https://2009.igem.org/Team:Freiburg_bioware/Project/FOS">Read more...</a>
-
       <td>We also thought of an alternative way of binding of the
+
    </td>
-
heterodimeric
+
-
Fok to the DNA avoiding the necessity of labelled oligos and their
+
-
binding proteins using the binding domain of a transcription factor as
+
-
a new adapter. We focused on the binding domain of the activator
+
-
protein-1 (AP-1) a crucial transcription factor implicated in numerous
+
-
cancers. The protein is composed by a series of dimers. Nine homologues
+
-
of the AP-1 leucine zipper region have been characterized, among them
+
-
c-Fos, c-Jun and semirational library-designed winning peptides FosW
+
-
and JunW. Via leucin zipper they interact among each other and with
+
-
their basic region they bind DNA.</td>
+
     </tr>
     </tr>
   </tbody>
   </tbody>
</table>
</table>
-
<a href="https://2009.igem.org/Team:Freiburg_bioware/Project/FOS">Read
+
 
-
more...</a>
+
 
<hr style="width: 100%; height: 2px;" />
<hr style="width: 100%; height: 2px;" />
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<h4><a name="Cloning_strategy"></a>Cloning
+
 
-
strategy</h4>
+
 
-
<a href="#">Read more...</a>
+
<h4><a name="Cloning_strategy"></a>Cloning strategy</h4>
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<hr style="width: 100%; height: 2px;" /></div>
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<table style="text-align: justify; width: 100%"; border="0" cellpadding="5" cellspacing="0">
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</div>
+
 
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<div class="cleared"></div>
+
    <td>
-
</div>
+
    Here we give an overview of the cloning strategies we used for creating a universal restriction endonuclease.
-
</div>
+
    Fusion proteins were generated according to the Freiburg Assembly standard RFC 25.
-
</div>
+
    <a href="https://2009.igem.org/wiki/index.php?title=Team:Freiburg_bioware/Project/clonestrat">Read more...</a>
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<div class="art-Post">
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<div class="art-Post-tc"></div>
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<div class="art-Post-bc"></div>
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<h2 class="art-PostHeaderIcon-wrapper"><a
+
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name="Literature_"></a> &nbsp;<img
+
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src="https://static.igem.org/mediawiki/2009/6/69/Freiburg09_PostHeader_tanne.png"
+
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alt="" style="width: 26px; height: 25px;" />
+
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Literature<span class="art-PostHeader"></span> </h2>
+
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<div class="art-PostContent">
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<div style="text-align: center;"></div>
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<div class="cleared"></div>
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<div class="art-Footer">
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<div class="art-Footer-text">
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<p>contact:&nbsp; <a
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href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a><br />
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</p>
</p>
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</div>
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</div>
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<div class="art-Footer-background"></div>
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<p>contact: <a href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a>;
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</div>
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<a href="mailto:freigem09@googlemail.com">kristian@biologie.uni-freiburg.de</a></p>
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Latest revision as of 08:32, 20 November 2009

FREiGEM



The goal of providing an universal restriction enzyme was approached with two design strategies. The first strategy evolves around novel protein fusion constructs combining the cleavage domain of the Type IIs restriction enzyme FokI with hapten binding anticalins, which are then guided to their target sites by a modified oligonucleotide. The second approach aimed at converting the Argonaute proteins from thermophilic organisms from an RNase to an DNase activity while accepting a DNA guide oligonucleotide. For the Fok-based strategy, several variations were tested. In both projects important milestones were reached. Most importantly, we demonstrated guided cleavage by one of our Fok-based fusion constructs and phage display of an Argonaute protein. In the following we introduce these two projects and then list the highlights of our work with links to detailed descriptions of each project part.
Both strategies rely on locating the universal restriction enzyme at the cleavage site with adapter or guide oligonucleotides. This is in contrast to previous designs which either use chemical linkage of an oligonucleotide to a nuclease or genetic fusion with a DNA binding domain. Previous fusion protein approaches have the conceptual disadvantage that for each target sequence a special protein has to be designed, expressed, purified, and stored. We only need to produce one protein. The oligonucleotide planning is aided by readily available PCR primer design programs and there are virtually no limits to define the cleavage site. Modified oligonucleotides can be ordered from many suppliers at low cost with short delivery times and easily can be stored long term. In in-vitro applications hybridization of the oligonucleotide is easily achieved also to double strand by heating and cooling as it is well known from PCR procedures. In case of the Argonaute proteins from thermophiles we can assume that they survive several cycles of heat dissociation and annealing. In case of the Fok we plan to introduce thermostability by directed evolution. In addition, other groups are working with oligonucleotides forming triple helices and their general hybridization with any sequence or with peptide nucleotide acids which provide higher stability and sneak in existing double helices. These technologies are compatible with our approach.
In both of our universal restriction enzyme strategies we do not cut the double strand but rather nick the stand opposite to our guide oligonucleotide. Thus, our guide oligonucleotide only needs to be added only in catalytic quantities. The nicking feature is already present in the Argonaute proteins. In the case of the universal Fok-based enzyme we use a heterodimer design combined with cleavage inactivating mutations for one monomer.

Details of the universal restriction enzyme based on Fok-Anticalin fusions
The restriction endonuclease FokI from Flavobacterium okeanokoites is a well studied protein. It consists of two domains, a DNA recognition domain and a DNA cleavage domain. Upon recognition of one target site and dimerization it cleaves the DNA nine bases apart from the recognition site. Several groups reported experiments to uncouple the cleavage and restriction domains of FokI and created a novel site-specific endonucleases by linking the cleavage domain to zinc finger proteins (Miller et al. 2007).
For our project we combined two previous research results and generated a Fok cleavage heterodimer comprising an enzymatically active and inactive monomer. For the catalytic active Fok partner, named Fok_a, as well as for the catalytic inactive Fok partner, Fok_i, the association interface was mutated to disfavor homodimerization and promote heterodimerization. In Fok_i additional amino acid exchanges led to the inactivation.
The two heterodimeric partners were fused to anticalins binding different adapter molecules. Fok_a is genetically fused to a digoxigenin-binding anticalin (DigA) and Fok_i to a fluorescein-binding anticalin (FluA). The adapter molecules digoxigenin and fluorescein are common modifications linked to oligonucleotides thus mediating the binding to the DNA site of interest. Two modifications allow for a better spatial control of the cleavage site. On the target site Fok_i and Fok_a constructs are brought into close proximity and can form a heterodimer. The inactive Fok domain will serve as an activator of the active Fok domain, which cuts one strand of the DNA. Our structural ‘3D’ models, indicate that Fok domains can be positioned in such a way that Fok_a will cleave the target DNA, and Fok_i would be directed towards the modified oligonucleotide. Different linkers were designed and fused between cleavage domain and the anticalin binding moieties to test for the optimal distance. In addition we generated a monomeric Fok fusion construct, to enable phage display and te4st a further setting requiring only one binding domain and hapten. Furthermore, we made a Fok cleavage domain fusion with a coiled coil based DNA binding domain, because we can combine these with existing light switchable inhibitors, which prevent DNA binding. As a result we will obtaina light switchable restriction enzyme.

Milestones
To reach our goal within the short given time frame we started several subprojects in parallel. Our subprojects listed here are defined along these projects.
Designing of the constructs was aided by extensive model building and analysis of the spatial orientation of the different proteins and oligonucleotides used. All designed and constructed parts feature full BioBrick compatibility and in addition allow for the construction of fusion proteins based on the RFC 25 (Freiburg) cloning standard. Cloning of the respective parts was followed by expression purification and analysis. Despite previous literature data our active Fok construct was toxic to cells when expressed and we tested periplasmic expression with export of the nascent polypeptide chain before folding.
In our experiments we addressed the following questions:
•    Structural Model building
•    Design of protein fusion parts
•    Cloning of anticalin Fok fusions
•    Cloning of a monomeric Fok construct and of a Jun/Fos directed Fok construct
•    Expression and purification of constructs
•    In vitro assays
•    In vivo assays
•    Phage Display of an Ago protein
•    Modeling of assembly and cleavage with differential equations
•    An international survey of laymen on synthetic biology

In short, we successfully worked on all aspects. Experiments to validate our approach in more compelx settings are ongoing.
The labs of Kristian Müller and Katja Arndt provided all technology and support for the project.


Schematic Model of the universal restriction enzymes based on
FokI and anticalins.

Structural model of the universal restriction enzymes based on FokI and anticalins.

Model of the catalytic cycle; hybridization - cleavage - temperature
promoted release.

Structure of an Ago protein, demonstrating guide oligonucleotide
mediated binding.

Modeling of the Enzyme Kinetics

For our modeling analyses we constructed various sets of differential equations describing protein-protein and protein-DNA interactions and the final cleavage.
Read more...

Modeling of the Enzyme Structure

Structural modeling was an initial step towards our planned universal restriction enzyme. Using molecular display software we arranged published crystal structures of FokI, anticalins and DNA. The arrangement was guided by superimposition with further structures of enzyme bound DNA. The modeling defined spatial requirements for linker lengths and positioning of modifications within oligonucleotides. Read more...

In vitro assays

After the cloning, expression and the purification of the Fok constructs we conducted several assays in order to to analyze the activity of the enzyme. To establish the assay and as a reference for activity we used wild type FokI. Binding of the modified nucleotides and enzymatic activity were tested with the Fok_i / Fok_a construct. Read more...

In vivo Assays

We demonstrated that modified guide oligonucleotides can be transfected in E. coli. Importantly, we showed that a M13 DNA hybridized with a guide oligonucleotide does not produce phages when transfectd in cells expressing Fok fusion proteins whereas the control M13 DNA does. Read more...

Protein Expression and Purification

Proteins were recombinantly expressed in E. coli strains BL21 or RV308. Purification was achieved by affinity chromatography utilizing the His-tag, Strep-tag, or the GST fusion. If needed, two affinity purifications due to two available tags were combined or a size exclusion chromatogrpahy was added. Read more...

AGO

The Argonaute proteins represent our second approach to generate universal restriction enzymes. These RNases provide already important features such as recognition of guide oligonucleotides and thermostability. We expressed and purified an AGO protein and showed residual DNase activtiy. Consequently, we started phage display of error prone PCR diversified AGO in order to convert the RNase in a DNase. We demonstratd AGO display, successfully completed two rounds of phage display and identified interesting mutants. Read more...

Ethics

We conducted an international survey on benefit and risk perception of Synthetic Biology by laymen, which was translated in 10 languages. Read more...

Fok Monomer

To ease production, purification, handling and analysis we cloned a monomeric Fok variant. In addition, this variant is better suited for phage display, which will allow us selection for improved properties. This protein comprises four functional subunits interspaced by two short and one long linker (anticalin-Fok_i-long_linker-Fok_a-anticalin). Read more...

Alternative way of binding: Jun/Fos

As an alternative way of binding of Fok to DNA we cloned the DNA binding domain (bZIP) of the activator protein-1 (AP-1). As light switching of coiled coils is established in our host lab, this will give us a light switchable restriction enzyme. Read more...

Cloning strategy

Here we give an overview of the cloning strategies we used for creating a universal restriction endonuclease. Fusion proteins were generated according to the Freiburg Assembly standard RFC 25. Read more...

contact: freigem09@googlemail.com; kristian@biologie.uni-freiburg.de