Difference between revisions of "Team:Marburg/Description"

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<div class="skipTarget" skipname="Golden Gate system"></div>
 
<p>
 
<p>
Golden Gate Assembly is a revolutionary cloning method published 10 years ago (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647">Engler <i>et al.</i>, 2008</a>). since its publication it became very popular giving rise to many assembly strategies based on this system. All systems published over the years offered improvements over previous publications. Nevertheless, although there are many systems available – often specialized for certain organisms or classes – the development by far is not at an end. We worked on a novel toolbox and developed an improved, highly flexible system focused on prokaryotes including a high number of parts characterized for the novel upcoming chassis <i>V. natriegens</i>.
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Golden Gate Assembly is a revolutionary cloning method published 10 years ago (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647">Engler <i>et al.</i>, 2008</a>). since its publication it became very popular giving rise to many assembly strategies based on this system. All systems published over the years offered improvements over previous publications. Nevertheless, although there are many systems available – often specialized for certain organisms or classes – the development by far is not at an end. We worked on a novel toolbox and developed an improved, highly flexible system focused on prokaryotes including a high number of parts characterized for the novel upcoming chassis <i>Vibrio natriegens</i>.
 
<br>
 
<br>
 
<br>
 
<br>
The innovation given by Golden Gate assembly was the introduction of type IIS restriction enzymes used for DNA digestion. A current workflow at that time consisted of DNA restriction, gel purification and finally ligation of the single parts. For restriction different type II restriction enzymes were taken making it important to check for compatible buffers and getting a six base pair scar after ligation. By introducing type IIS restriction enzymes into this workflow the system was highly improved. This new method offered many advantages like I) an independent enzyme recognition site that is removed after restriction, II) restriction and ligation in one pot III) directional assembly by conscious design of the cleavage sequences IV) no buffer incompatibilities due to the usage of one enzyme cutting at several spots and still producing different cleavage sequences (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647">Engler <i>et al.</i>, 2008</a>) (Figure will be added…).<br>
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The innovation given by Golden Gate assembly was the introduction of type IIS restriction enzymes used for DNA digestion. A current workflow at that time consisted of DNA restriction, gel purification and finally ligation of the single parts. For restriction different type II restriction enzymes were taken making it important to check for compatible buffers and getting a six base pair scar after ligation. By introducing type IIS restriction enzymes into this workflow the system was highly improved. This new method offered many advantages like I) an independent enzyme recognition site that is removed after restriction, II) restriction and ligation in one pot III) directional assembly by conscious design of the cleavage sequences IV) no buffer incompatibilities due to the usage of one enzyme cutting at several spots and still producing different cleavage sequences (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003647">Engler <i>et al.</i>, 2008</a>).
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<br>
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<figure align="middle">
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<div class="imageContainer1x2">
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<img src="https://static.igem.org/mediawiki/2018/0/03/T--Marburg--OrisGraph.jpeg">
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</div>
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<figcaption>
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<b>Figure 1: Golden Gate assembly<br></b>
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Image description
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</figcaption>
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</figure>
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<br>
 
Based on this, many new systems arose offering the possibility of building complex constructs out of single genetic parts in a fast, simple and flexible way. Full libraries of standardized genetic parts were developed enabling the user to build full combinatorial libraries. The first system developed was the modular cloning system (MoClo) offering a new hierarchical cloning method of defined biological parts (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765">Weber <i>et al.</i>, 2011</a>) and consequently setting a new standard over the years.<br>
 
Based on this, many new systems arose offering the possibility of building complex constructs out of single genetic parts in a fast, simple and flexible way. Full libraries of standardized genetic parts were developed enabling the user to build full combinatorial libraries. The first system developed was the modular cloning system (MoClo) offering a new hierarchical cloning method of defined biological parts (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765">Weber <i>et al.</i>, 2011</a>) and consequently setting a new standard over the years.<br>
Here we summarize some Golden Gate based systems and compare their advantages and disadvantages.
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Here we summarize some Golden Gate based systems in a chronological manner looking at the improvements over the years and compare their advantages and disadvantages.
 
</p>
 
</p>
 
 
<figure>
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<div class="imageContainer1x2">
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<img src="">
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</div>
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<figcaption>
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<b>Figure 1: Golden Gate assembly<br></b>
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Image description
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</figcaption>
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</figure>
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<div class="collapsible">
 
<div class="collapsible">
<div class="btn_expand">Modular Cloning (MoClo)</div>
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<div class="btn_expand">Modular Cloning (MoClo) (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016765">Weber <i>et al.</i>, 2011</a>)</div>
 
<div class="content">
 
<div class="content">
 
<div class="skipTarget" skipname="Modular Cloning"></div>
 
<div class="skipTarget" skipname="Modular Cloning"></div>
 
 
 
<p>
 
<p>
The modular cloning system was the first one proposing a standard for Golden Gate based assembly. This toolbox offers 5 types of modules designed mainly for eukaryotes. The modules are stored in level 0 acceptor plasmids derived from the pUC19 backbone with a spectinomycin resistance and a LacZα cassette as dropout for blue/white screening. Custom level 0 plasmids are assembled by flanking the sequences with BpiI recognition sites and setting a single restriction-ligation-reaction with the correspondent plasmid. Up to 5 level 0 modules are assembled in an acceptor plasmid with ampicillin resistance and a LacZα-dropout by restriction with BsaI to transcription units. 6 transcription units can be assembled using BpiI into level 2 multigene constructs containing a kanamycin resistance and a Cred-dropout. Alternatively Esp3I can be use to transfer the constructs to intermediary levels to reach higher levels for assembly of bigger constructs.
+
The modular cloning system was the first one proposing a standard for Golden Gate based assembly. This toolbox offers 5 types of modules designed mainly for eukaryotes. The modules are stored in level 0 acceptor plasmids derived from the pUC19 backbone with a spectinomycin resistance and a LacZα cassette as dropout for blue/white screening. Custom level 0 plasmids are assembled by flanking the sequences with BpiI recognition sites and setting a single restriction-ligation-reaction with the correspondent plasmid. Up to 5 level 0 modules are assembled in an acceptor plasmid with ampicillin resistance and a LacZα-dropout by restriction with BsaI to transcription units. 6 transcription units can be assembled using BpiI into level 2 multigene constructs containing a kanamycin resistance and a Cred-dropout. Alternatively Esp3I can be use to transfer the constructs to intermediary levels to reach higher levels for assembly of bigger constructs.<br>
 +
This toolbox is best for constructs up to level 2 plasmids. Higher levels can be reached through intermediary levels but need two restriction enzymes for the assembly. Furthermore it is important to know in which level the current constructs stand to avoid messing up the acceptor plasmids for subsequent levels.
 
</p>
 
</p>
 
</div>
 
</div>
 
</div>
 
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<div class="collapsible">
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<div class="btn_expand">Golden Braid (<a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021622">Sarrion-Perdigones <i>et al.</i>, 2011</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="Golden Braid"></div>
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<p>
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The Golden Braid system was mainly developed with focus on plants. It picks up the idea of the MoClo system and improves it offering a looping system for reaching higher levels of assembly. Level 0 modules are stored in entry plasmids surrounded by BsaI restriction sites containing an ampicillin resistance and a LacZα dropout. This modules are assembled to transcription units in one of two level α entry plasmids with kanamycin resistance and a LacZα dropout. Here the transcription units are surrounded by BsmBI restriction sites. The transcription units can subsequently be transferred into level Ω entry plasmids with spectinomycin resistance and BsaI restriction sites surrounding the multigene constructs. Every level α plasmid is a precursor of a level Ω plasmid and vise versa. This way the system allows to loop over the plasmids building in theory infinite bigger constructs doubling their size on every round.<br>
 +
This system makes the assembly of higher levels much simpler using only 1 restriction enzyme. Nevertheless the plasmids’ size is just doubled from level to level.
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</p>
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<div class="collapsible">
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<div class="btn_expand">Golden Braid 2.0 (<a href="http://www.plantphysiol.org/content/162/3/1618">Sarrion-Perdigones <i>et al.</i>, 2013</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="Golden Braid 2.0"></div>
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<p>
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The Golden Braid 2.0 system is an improvement of its prior system. Some minor changes were introduced to make the system simpler and more versatile. The basic modules were defined and classified in 11 classes building optional subclasses e.g. protein tags. The level 0 modules are stored in a universal vector (pUPD) surrounding the sequences with BsaI and BtgZI recognition sites cutting on the same position. The current acceptor plasmids α and Ω have been supplemented by reverse plasmids allowing the inversion of single transcription units.  The fusion sites were simplified by merging unnecessary ones. ‘Superparts’ were introduced consisting of pre-assembled modules to reduce the number of fragments needed for the assembly.<br>
 +
This changes do not create a completely new toolbox but complements the system with some handy facilities improving it notably. However the system is still limited to a duplication of the plasmids size from level to level.
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</p>
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<div class="collapsible">
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<div class="btn_expand">MoClo – YTK (<a href="https://doi.org/10.1021/sb500366v">Lee <i>et al.</i>, 2015</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="The yeast tollkit"></div>
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<p>
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The yeas toolkit, also known as the Dueber toolbox, offers a Golden Gate based system adapted for yeast. The basic level 0 parts are classified in 8 types with optional subtypes. New basic parts are assembled into entry plasmids by restriction with BsmBI. For building level 1 cassettes at least 8 parts are assembled by a restriction-ligation step using BsaI. The innovation of this toolkit compared to the previously reviewed is the use of connector sequences for level 1 and higher assembly steps. This way plasmids for yeast can be built <i>de novo</i> without the need of a defined backbone. Furthermore they integrated a method for simple chromosomal integration by lenearization of the plasmids with NotI. On top of this the connectors can be used as homology sequences e.g. for ligation-independent cloning, Gibson assembly, ligase cycling reaction or yeast <i>in vivo</i> assembly. Although this toolkit focuses mainly on yeast it can be used for working with <i>E. coli</i> as well. Unfortunately this toolkit does not arrange to any standard becoming incompatible with other toolkits.
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</p>
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</div>
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<div class="collapsible">
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<div class="btn_expand">PhytoBrick (<a href="https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/nph.13532">Patron <i>et al.</i>, 2015</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="PhytoBrick"></div>
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<p>
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The PhytoBrick system does not offer another genetic toolbox but a wide standard compatible with popular systems like GB 2.0 and MoClo aiming to create a standard focused primary on plant engineering efforts. The iGEM competition already accepted it as an standard and offers support for building parts designed for plants, yeast and bacteria. This system proposes 12 defined fusion sites applicable for the different genetic modules. The fusion sites are divided into three major classes for promoter parts, transcribed regions and terminator parts. This classes are divided into subclasses giving the flexibility to use optional modules like tags, promoter regulators and enhancer regions. The system also proposes two types of universal acceptor plasmids (UAPs) derived from the pSB1C3 plasmid where level 0 modules can be inserted by a single restriction-ligation step with BpiI or BsmBI respectively.
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</p>
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<div class="collapsible">
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<div class="btn_expand">CIDAR MoClo (<a href="https://doi.org/10.1021/acssynbio.5b00124">Iverson <i>et al.</i>, 2016</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="CIDAR MoClo"></div>
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<p>
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The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library represents the first toolbox adapted for bacteria focused on <i>E. coli</i>. It offers 95 genetic modules for bacteria and proposes a modified protocol reducing the reaction time of the assembly steps from 5 hours to just 90 minutes. The toolkit provides a series of level 0 plasmids derived from the iGEM pSB1A2 backbone. Four modules are assembled into a series of level 1 destination plasmids derived from pSB1K3 to transcription units using BsaI. In a next step this transcription units can be assembled to multigene modules by restriction with BbsI. Up to 4 transcription units can be assembled to a level 2 multigene construct.
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</p>
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<div class="collapsible">
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<div class="btn_expand">EcoFlex (<a href="https://doi.org/10.1021/acssynbio.6b00031">Moore <i>et al.</i>, 2016</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="EcoFlex"></div>
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<p>
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EcoFlex is a toolbox specialized on the use with <i>E. coli</i> that offers a wide variety of modules. It features a library of constitutive promoters, T7 expressions, RBS variations, synthetic terminators, protein purification tags and fluorescence proteins characterized <i>in vivo</i> and <i>in vitro</i>. Furthermore it is compatible with MoClo, Golden Braid 2.0 and PhytoBrick standard.<br>
 +
One unique feature of this kit is the ‘entry vector’ for ORFs. The parts are flanked by NdeI and BamHI sites again flanked by BsaI restriction sites. The NdeI sites provide the fusion sites for Golden Gate assembly without addition of amino acids to the coded protein and in combination with BamHI additionally  providing compatible transfer to the commercial available pET vectors. If NdeI or BamHI are present in the coded protein NcoI and BglII can be used respectively for the assembly.<br>
 +
The second unique feature of EcoFlex is the inclusion of a secondary module. Here level 2 transcription units (TUs) can be subcloned into a corresponding secondary module using compatible BpiI sites located upstream the primary MoClo BsmBI sites. This way a module of multiple TUs can be ligated into a secondary module containing further TUs. The introduction of this feature offers a simplified and faster method for combinatorial pathway optimization.
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</p>
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<div class="collapsible">
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<div class="btn_expand">Loop assembly (<a href="https://www.biorxiv.org/content/early/2018/01/18/247593">Pollak <i>et al.</i>, 2018</a>)</div>
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<div class="content">
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<div class="skipTarget" skipname="Loop assembly"></div>
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<p>
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Loop assembly provides a new toolbox designed for plant engineering purposes. It contains a series of 8 plasmids derived from pGreenII backbone divided in two sets to loop over several levels of assembly. Level 0 modules are assembled into odd receiver plasmids (pOdd) with a kanamycin resistance by restriction with BsaI. Four odd level transcription units are assembled into one even level receiver plasmid (pEven) with a spectinomycin resistance by restriction with SapI. The system can be elevated to higher levels by looping over odd and even receiver plasmids exponentially by the factor of 4. For the case that less than 4 modules are needed spacer plasmids are offered of 200bp random DNA free of BsaI or SapI recognition sites to close the gaps. The level 0 parts’ fusion sites are designed using the PhytoBrick standard previously reviewed and are therefore compatible with GB 2.0 and MoClo. A further improvement of this system is the use of unique nucleotide sequences (UNS) set between single transcription units. This UNS allow highly efficient PCR amplification of single functional units and allow the assembly into the plasmids by overlap assembly methods like Gibson assembly as well.<br>
 +
Although this is an improved and very simple but useful system the usage of SapI delivers a 3 bp fusion site restricting the number of possible fusion sites to a lower number and simultaneously downgrading compatibility with previous systems.
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</p>
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</div>
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<div class="collapsible">
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<div class="btn_expand">Summary</div>
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<div class="content">
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<div class="skipTarget" skipname="Genetic toolkits summary"></div>
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<table style="width:100%">
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<tr>
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<th>Toolkit</th>
 +
<th>MoClo</th>
 +
<th>Golden Braid</th>
 +
<th>Golden Braid 2.0</th>
 +
<th>MoClo – YTK</th>
 +
<th>PhytoBrick</th>
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<th>CIDAR MoClo</th>
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<th>EcoFlex</th>
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<th>Loop Assembly</th>
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</tr>
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<tr>
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<th>Year of publication</th>
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<td>2011</td>
 +
<td>2011</td>
 +
<td>2013</td>
 +
<td>2015</td>
 +
<td>2015</td>
 +
<td>2015</td>
 +
<td>2016</td>
 +
<td>2018</td>
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</tr>
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<tr>
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<th>Restriction sites</th>
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<td>BsaI, BpiI (Esp3 for intermediary levels)</td>
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<td>BsaI, BsmBI</td>
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<td>BsaI, BsmBI (BtgZI as BsaI alternative)</td>
 +
<td>BsaI, BsmBI (NotI for chromosomal integration)</td>
 +
<td>BsaI, BsmBI (BpiI for compatibility with MoClo)</td>
 +
<td>BsaI, BbsI</td>
 +
<td>BsaI, BsmBI (NdeI + BamHI for secondary modules)</td>
 +
<td>BsaI, SapI</td>
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</tr>
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 +
<tr>
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<th>Dropouts for negative selection</th>
 +
<td>LacZα, CRed</td>
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<td>LacZα</td>
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<td>LacZα</td>
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<td>sfGFP (only lvl 0)</td>
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<td>Depends on the system respectively</td>
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<td>LacZα</td>
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<td>LacZα, RFP</td>
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<td>LacZα</td>
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</tr>
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<tr>
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<th>Resistances</th>
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<td>Spectinomycin → ampicillin → kanamycin</td>
 +
<td>Ampicillin → kanamycin → spectinomycin</td>
 +
<td>Ampicillin → kanamycin → spectinomycin</td>
 +
<td>chloramphenicol → ampicillin → kanamycin</td>
 +
<td>Chloramphenicol, ampicillin, kanamycin, spectinomycin</td>
 +
<td>Ampicillin → kanamycin</td>
 +
<td>Chloramphenicol → β-lactamase</td>
 +
<td>Kanamycin → spectinomycin</td>
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</tr>
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<tr>
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<th>Target organisms</th>
 +
<td>Mainly eukaryotes</td>
 +
<td>Plants</td>
 +
<td>Plants</td>
 +
<td>Yeast</td>
 +
<td>Plants</td>
 +
<td>Bacteria</td>
 +
<td>E. coli</td>
 +
<td>Plants</td>
 +
</tr>
 +
 +
<tr>
 +
<th>Special features</th>
 +
<td>First Golden Gate Assembly standard</td>
 +
<td>Theoretically infinite looping over two sets of plasmids and levels</td>
 +
<td>Flexible direction inversion of single TUs during assembly steps, introduction of classes and subclasses</td>
 +
<td>Connector-based de novo plasmid assembly for yeast, chromosomal integration, </td>
 +
<td>Not a toolbox but a proposal for a standard widely compatible with popular systems, accepted standard for the iGEM competition</td>
 +
<td>First system focused on bacteria, enhanced protocol for Golden Gate reaction in 90 minutes</td>
 +
<td>Widely versatile toolbox, adapted for E. coli, designed for circuit design, combinatorial pathway optimization, recombinant protein purification</td>
 +
<td>Simple usage, wide compatibility due to PhytoBrick standard, usage of UNS</td>
 +
</tr>
 +
 +
<tr>
 +
<th>Enhancement per level</th>
 +
<td>6 TUs, but capped at level 3</td>
 +
<td>2n</td>
 +
<td>2n</td>
 +
<td></td>
 +
<td>Depends on the system respectively</td>
 +
<td>4n</td>
 +
<td>Up to 5 TUs on level 2 and up to 20 on level 3</td>
 +
<td>4n</td>
 +
</tr>
 +
 +
<tr>
 +
<th>Number of parts</th>
 +
<td>95</td>
 +
<td></td>
 +
<td>94</td>
 +
<td>96</td>
 +
<td></td>
 +
<td>93</td>
 +
<td>78</td>
 +
<td>58</td>
 +
</tr>
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</table>
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</div>
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</div>
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  <div class="btn_expand"><div class="skipTarget" skipname="Part Collection"></div>
 
    Part Collection</div>
 
  <div class="content">
 
<div style="width:100%">
 
<img style="display:block; margin:0 auto; width:50%; max-width:1080px" src="https://static.igem.org/mediawiki/2018/3/36/T--Marburg--PartCollectionLogo.png">
 
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Revision as of 19:56, 17 October 2018

Description

you better start swimmin'
Or you'll sink like a stone
For the times they are a-changin'

--Bob Dylan


The last years have seen a renaissance of research into V. natriegens, the world’s fastest growing bacterium. Vibrio natriegens has a lot of potential to become the next universally applicable chassis organism, challenging E. coli’s dominance. To fully realize this potential, several steps have to be taken. We intended to contribute to this coup d’état by providing the research community with open source strains, tailored specifically to cloning, protein expression and interaction. Additionally, we conducted basic microbial research, establishing procedures and protocols for cloning and molecular biology research.

Metabolic engineering relies heavily on iterative testing. To shorten the duration of these iterations means to enable broader and more in-depth acquisition of data.

Traditional cloning methods are limited by their rigidity and the time it takes to assemble constructs from multiple fragments. Recently, cloning methods based on type IIs restriction enzymes have addressed these issues. The synergy generated by combining these with V. natriegens would result in the world's fastest cloning platform, the Marburg Collection.

Part Collection
Genetic toolboxes, a review

Golden Gate Assembly is a revolutionary cloning method published 10 years ago (Engler et al., 2008). since its publication it became very popular giving rise to many assembly strategies based on this system. All systems published over the years offered improvements over previous publications. Nevertheless, although there are many systems available – often specialized for certain organisms or classes – the development by far is not at an end. We worked on a novel toolbox and developed an improved, highly flexible system focused on prokaryotes including a high number of parts characterized for the novel upcoming chassis Vibrio natriegens.

The innovation given by Golden Gate assembly was the introduction of type IIS restriction enzymes used for DNA digestion. A current workflow at that time consisted of DNA restriction, gel purification and finally ligation of the single parts. For restriction different type II restriction enzymes were taken making it important to check for compatible buffers and getting a six base pair scar after ligation. By introducing type IIS restriction enzymes into this workflow the system was highly improved. This new method offered many advantages like I) an independent enzyme recognition site that is removed after restriction, II) restriction and ligation in one pot III) directional assembly by conscious design of the cleavage sequences IV) no buffer incompatibilities due to the usage of one enzyme cutting at several spots and still producing different cleavage sequences (Engler et al., 2008).

Figure 1: Golden Gate assembly
Image description

Based on this, many new systems arose offering the possibility of building complex constructs out of single genetic parts in a fast, simple and flexible way. Full libraries of standardized genetic parts were developed enabling the user to build full combinatorial libraries. The first system developed was the modular cloning system (MoClo) offering a new hierarchical cloning method of defined biological parts (Weber et al., 2011) and consequently setting a new standard over the years.
Here we summarize some Golden Gate based systems in a chronological manner looking at the improvements over the years and compare their advantages and disadvantages.

Modular Cloning (MoClo) (Weber et al., 2011)

The modular cloning system was the first one proposing a standard for Golden Gate based assembly. This toolbox offers 5 types of modules designed mainly for eukaryotes. The modules are stored in level 0 acceptor plasmids derived from the pUC19 backbone with a spectinomycin resistance and a LacZα cassette as dropout for blue/white screening. Custom level 0 plasmids are assembled by flanking the sequences with BpiI recognition sites and setting a single restriction-ligation-reaction with the correspondent plasmid. Up to 5 level 0 modules are assembled in an acceptor plasmid with ampicillin resistance and a LacZα-dropout by restriction with BsaI to transcription units. 6 transcription units can be assembled using BpiI into level 2 multigene constructs containing a kanamycin resistance and a Cred-dropout. Alternatively Esp3I can be use to transfer the constructs to intermediary levels to reach higher levels for assembly of bigger constructs.
This toolbox is best for constructs up to level 2 plasmids. Higher levels can be reached through intermediary levels but need two restriction enzymes for the assembly. Furthermore it is important to know in which level the current constructs stand to avoid messing up the acceptor plasmids for subsequent levels.

The Golden Braid system was mainly developed with focus on plants. It picks up the idea of the MoClo system and improves it offering a looping system for reaching higher levels of assembly. Level 0 modules are stored in entry plasmids surrounded by BsaI restriction sites containing an ampicillin resistance and a LacZα dropout. This modules are assembled to transcription units in one of two level α entry plasmids with kanamycin resistance and a LacZα dropout. Here the transcription units are surrounded by BsmBI restriction sites. The transcription units can subsequently be transferred into level Ω entry plasmids with spectinomycin resistance and BsaI restriction sites surrounding the multigene constructs. Every level α plasmid is a precursor of a level Ω plasmid and vise versa. This way the system allows to loop over the plasmids building in theory infinite bigger constructs doubling their size on every round.
This system makes the assembly of higher levels much simpler using only 1 restriction enzyme. Nevertheless the plasmids’ size is just doubled from level to level.

The Golden Braid 2.0 system is an improvement of its prior system. Some minor changes were introduced to make the system simpler and more versatile. The basic modules were defined and classified in 11 classes building optional subclasses e.g. protein tags. The level 0 modules are stored in a universal vector (pUPD) surrounding the sequences with BsaI and BtgZI recognition sites cutting on the same position. The current acceptor plasmids α and Ω have been supplemented by reverse plasmids allowing the inversion of single transcription units. The fusion sites were simplified by merging unnecessary ones. ‘Superparts’ were introduced consisting of pre-assembled modules to reduce the number of fragments needed for the assembly.
This changes do not create a completely new toolbox but complements the system with some handy facilities improving it notably. However the system is still limited to a duplication of the plasmids size from level to level.

MoClo – YTK (Lee et al., 2015)

The yeas toolkit, also known as the Dueber toolbox, offers a Golden Gate based system adapted for yeast. The basic level 0 parts are classified in 8 types with optional subtypes. New basic parts are assembled into entry plasmids by restriction with BsmBI. For building level 1 cassettes at least 8 parts are assembled by a restriction-ligation step using BsaI. The innovation of this toolkit compared to the previously reviewed is the use of connector sequences for level 1 and higher assembly steps. This way plasmids for yeast can be built de novo without the need of a defined backbone. Furthermore they integrated a method for simple chromosomal integration by lenearization of the plasmids with NotI. On top of this the connectors can be used as homology sequences e.g. for ligation-independent cloning, Gibson assembly, ligase cycling reaction or yeast in vivo assembly. Although this toolkit focuses mainly on yeast it can be used for working with E. coli as well. Unfortunately this toolkit does not arrange to any standard becoming incompatible with other toolkits.

PhytoBrick (Patron et al., 2015)

The PhytoBrick system does not offer another genetic toolbox but a wide standard compatible with popular systems like GB 2.0 and MoClo aiming to create a standard focused primary on plant engineering efforts. The iGEM competition already accepted it as an standard and offers support for building parts designed for plants, yeast and bacteria. This system proposes 12 defined fusion sites applicable for the different genetic modules. The fusion sites are divided into three major classes for promoter parts, transcribed regions and terminator parts. This classes are divided into subclasses giving the flexibility to use optional modules like tags, promoter regulators and enhancer regions. The system also proposes two types of universal acceptor plasmids (UAPs) derived from the pSB1C3 plasmid where level 0 modules can be inserted by a single restriction-ligation step with BpiI or BsmBI respectively.

CIDAR MoClo (Iverson et al., 2016)

The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library represents the first toolbox adapted for bacteria focused on E. coli. It offers 95 genetic modules for bacteria and proposes a modified protocol reducing the reaction time of the assembly steps from 5 hours to just 90 minutes. The toolkit provides a series of level 0 plasmids derived from the iGEM pSB1A2 backbone. Four modules are assembled into a series of level 1 destination plasmids derived from pSB1K3 to transcription units using BsaI. In a next step this transcription units can be assembled to multigene modules by restriction with BbsI. Up to 4 transcription units can be assembled to a level 2 multigene construct.

EcoFlex is a toolbox specialized on the use with E. coli that offers a wide variety of modules. It features a library of constitutive promoters, T7 expressions, RBS variations, synthetic terminators, protein purification tags and fluorescence proteins characterized in vivo and in vitro. Furthermore it is compatible with MoClo, Golden Braid 2.0 and PhytoBrick standard.
One unique feature of this kit is the ‘entry vector’ for ORFs. The parts are flanked by NdeI and BamHI sites again flanked by BsaI restriction sites. The NdeI sites provide the fusion sites for Golden Gate assembly without addition of amino acids to the coded protein and in combination with BamHI additionally providing compatible transfer to the commercial available pET vectors. If NdeI or BamHI are present in the coded protein NcoI and BglII can be used respectively for the assembly.
The second unique feature of EcoFlex is the inclusion of a secondary module. Here level 2 transcription units (TUs) can be subcloned into a corresponding secondary module using compatible BpiI sites located upstream the primary MoClo BsmBI sites. This way a module of multiple TUs can be ligated into a secondary module containing further TUs. The introduction of this feature offers a simplified and faster method for combinatorial pathway optimization.

Loop assembly (Pollak et al., 2018)

Loop assembly provides a new toolbox designed for plant engineering purposes. It contains a series of 8 plasmids derived from pGreenII backbone divided in two sets to loop over several levels of assembly. Level 0 modules are assembled into odd receiver plasmids (pOdd) with a kanamycin resistance by restriction with BsaI. Four odd level transcription units are assembled into one even level receiver plasmid (pEven) with a spectinomycin resistance by restriction with SapI. The system can be elevated to higher levels by looping over odd and even receiver plasmids exponentially by the factor of 4. For the case that less than 4 modules are needed spacer plasmids are offered of 200bp random DNA free of BsaI or SapI recognition sites to close the gaps. The level 0 parts’ fusion sites are designed using the PhytoBrick standard previously reviewed and are therefore compatible with GB 2.0 and MoClo. A further improvement of this system is the use of unique nucleotide sequences (UNS) set between single transcription units. This UNS allow highly efficient PCR amplification of single functional units and allow the assembly into the plasmids by overlap assembly methods like Gibson assembly as well.
Although this is an improved and very simple but useful system the usage of SapI delivers a 3 bp fusion site restricting the number of possible fusion sites to a lower number and simultaneously downgrading compatibility with previous systems.

Summary
Toolkit MoClo Golden Braid Golden Braid 2.0 MoClo – YTK PhytoBrick CIDAR MoClo EcoFlex Loop Assembly
Year of publication 2011 2011 2013 2015 2015 2015 2016 2018
Restriction sites BsaI, BpiI (Esp3 for intermediary levels) BsaI, BsmBI BsaI, BsmBI (BtgZI as BsaI alternative) BsaI, BsmBI (NotI for chromosomal integration) BsaI, BsmBI (BpiI for compatibility with MoClo) BsaI, BbsI BsaI, BsmBI (NdeI + BamHI for secondary modules) BsaI, SapI
Dropouts for negative selection LacZα, CRed LacZα LacZα sfGFP (only lvl 0) Depends on the system respectively LacZα LacZα, RFP LacZα
Resistances Spectinomycin → ampicillin → kanamycin Ampicillin → kanamycin → spectinomycin Ampicillin → kanamycin → spectinomycin chloramphenicol → ampicillin → kanamycin Chloramphenicol, ampicillin, kanamycin, spectinomycin Ampicillin → kanamycin Chloramphenicol → β-lactamase Kanamycin → spectinomycin
Target organisms Mainly eukaryotes Plants Plants Yeast Plants Bacteria E. coli Plants
Special features First Golden Gate Assembly standard Theoretically infinite looping over two sets of plasmids and levels Flexible direction inversion of single TUs during assembly steps, introduction of classes and subclasses Connector-based de novo plasmid assembly for yeast, chromosomal integration, Not a toolbox but a proposal for a standard widely compatible with popular systems, accepted standard for the iGEM competition First system focused on bacteria, enhanced protocol for Golden Gate reaction in 90 minutes Widely versatile toolbox, adapted for E. coli, designed for circuit design, combinatorial pathway optimization, recombinant protein purification Simple usage, wide compatibility due to PhytoBrick standard, usage of UNS
Enhancement per level 6 TUs, but capped at level 3 2n 2n Depends on the system respectively 4n Up to 5 TUs on level 2 and up to 20 on level 3 4n
Number of parts 95 94 96 93 78 58

Strain Engineering
By creating strains suited for diverse applications, we accelerate your research!
Methods
We took a new procedure to create genomic modifications for creation of new lab strains.

Natural Competence

What happened with the genetic Information when cells lysis? The DNA will be released to the environment. But can it be reused there? Or is the hole information lost? Under certain circumstances other cells can take up the free DNA. This ability of cells is called competence. A special kind is the natural competence. These are bacteria that have genes in their genome encoded for proteins involved in the DNA recognition and uptake and in the integration in the genome.

In 1928 natural competence was first observed by Frederick Griffith even if he did not understand the genetics behind (Griffith et al.1928) . He already recognized that information can be transferred from one bacterium to another and thus he set the starting point of a big era to understand the DNA exchange. Thanks many more great scientists researched in this area, we now know alot about natural competence and the ability of some bacteria to take up free DNA. However, as in almost every research, there are still many open questions.

One of the most famous Vibrio species is Vibrio cholera. This bacterial species triggers the disease called cholera, which is well known. However, this bacterium is like many other vibrio species also interesting for many other genetic features. When you think about how he has achieved his pathogenic properties, it come into your mind that this bacterium is maybe capable to do extensive gene transfer. What we know is, that the most Vibrio species are natural competence, that means that these organism can take up linear DNA from the environment und this genetic material can used for nutrients, to repair the genome or to promote horizontal gene transfer when integrated it into their genome by homologous recombination (Antonova et al.2015) . There was identified multiple environmental cues sufficient to induce natural transformation. For the most Vibrio species the phenomenon of natural transformation is induced by Chitin (Meibom et al.2005) . In the natural aquatic environment of for example V. cholera there are many chitinous shells of crustacean zooplankton which induces the expression of the competence regulator Tfox (Meibom et al.2005).
For natural transformation several steps are necessary: At first the DNA has to be transported into the cell. After the DNA is bound to the cell, the Type IV Pillus (T4P) picks it up and transported it through secretion pores in the outer membrane into the periplasm. From there the DNA is translocated across the membrane by using a conserved membrane pore into the cytoplasm. If the DNA is arrived in the cell, it can be integrated by homologous recombination (Mellet al.2014).
This process is dependent on some cellular structures, including type IV Pilli or related pseudopilli and many competence-related proteins and various regulator systems. The regulator Tfox activated the Type VI secretion systems (T6SSs) and that allows bacteria to inject directly into prey cell membranes or cytoplasm. It is a nanomachine that have many functions, one is a part of the natural competence regulon. Although much process has been made in this area, the molecular details of natural competence is not understood completely (Wu et al.2015).

Is Vibrio natriegens - a member of the vibrio species - natural competent, too? At the moment it is not so much known about the functionality of this process in Vibrio natriegens. However, it was discovered that it is natural competent and the Tfox regulates this property. But, it is not clear how Tfox is induced. It has been shown that in V. natriegens it is not induced by chitin as is the case with most Vibrio species. It was shown that you can express Tfox on an IPTG-inducible plasmid and there you get a high rate of natural transformation. In 2017 the group of Ankur B. Dalia showed that it was possible to integrate a linear PCR product with an antibiotic cassette that replaces the gene encoding the DNA endonuclease Dns in V. natriegens by using natural transformation. Around 1-10% of the population had integrated the transforming DNA (Dalia et al.2017) this is the highest rate of transformation in competent species (Lorenz et al.1994) . Thus it was shown that a natural transformation can be well used to perform a scarfree genome editing (Dalia et al.2017).

A big advantage of the method is that it is possible to perform many genome changes together, where you only need one antibiotics cassette. Therefore cells are incubated with a selected and an unselected product. The selected product had to contain an antibiotics cassette and the unselected product is used for the scarless genome editing of interest at the desired locus. This was also shown with a remarkably efficient up to 80% in V. natriegens (Daliaet al.2017) . MuGENT can be used to rapidly generate V. natriegens strains with several large, scarless genome edits without off- targeting effects at high efficiency.That can be used to prepare highly complex mutant population. Many methods are described for modifying the genome of bacteria, but the great advantage of MuGENT is that it is not limited to small chances. You can efficiently swap, insert or remove whole units. In the future natural transformation could be utilized in combination with genome engineering methods like MAGE and CAGE, which rely on electroporation for E.coli, that could allow more rapid genome designs, such as complete codon reassignments.

The Flp/frt system

There are several methods available to manipulate bacterial chromosomes. As the strain engineering subgroup, we had to decide which kind of strategy to use. Since iGEM means a lot of work in a very short time, we decided to look up for a method that further accelerates our project. We could use common plasmid based methods that rely on selection for plasmid excision followed by its loss. For Gram-negative bacteria these strategy often relies on counter selectable levan-sucrase enzyme, encoded by sacB (Donnenberg & Kaper 1991) . The excision can restore the genotype before insertion or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. The disadvantage of this method is that it is time-consuming. First, you need to clone a plasmid and then horizontal gene transfer from a donor to V. natriegens would be necessary. Additionally, the excision of the plasmid can lead to the pre-insertion genotype.
One of the big advantages of Vibrio species is their ability to take up free DNA from their environment by natural competence (Blokesch et al. 2005) , (Wollenberg et al. 2010) . Using this skill, plasmid independent genomic modification with linear DNA fragments can be performed in V. natriegens as well. To select for clones, which integrated those linear fragments at least one fragment containing an antibiotics resistance cassette is needed (Hayes et al. 2017) .
It would be a big disadvantage to create strains not compatible to some of your plasmids because of the antibiotics resistances incompatibility. The solution is, to design those antibiotics cassettes in a way they can be removed after integration. It was shown, that genomic modifications using recombinase-based methods like the Cre/Lox system are possible in V. natriegens (Hesek et al. 2016) . A similar method is the FLP/FRT system. FLP is a protein of the integrase family recombinases derived from the yeast Saccharomyces cerevisiae (Landy et al. 1986) , (Sadowski 1995)) . The recombinases of this family are sharing conserved amino acids that are responsible for the mechanisms of strand cleavage and ligation by the integrase members (Chen et al. 1990) , (Pan & Sadowski 1992) .
The FLP recognition target contains an eight bp asymmetric core that is surrounded by two 13-bp symmetric elements (5'GAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'). The recombinase attaches to the top or bottom strand next to the core region, cleaves the DNA and promotes efficient recombination between two FRT recombination sites (Guarascio et al. 1982).
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Figure 1:

In general, Flp mediated recombination between these sites can lead to insertion, excision, inversion, and translocation of DNA, depending on the direction of the FRT sites (Figure 1). Using FRT sites that are oriented in the same direction, leads to Flp mediated deletion of the region in-between. (Sadowski 1995). This system works in Vibrio species like V. cholera or V. fischeri for rapid genetic modifications (Blokesch 2012) .
In order to establish a new and rapid method for genomic modifications in V. natriegens, we will use the FLP/FRT system to create our strains.

Integration
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Some content about Integration
Strains
We established three new strains derived from the original wildtype strain as chassis organisms for cloning, protein expression and protein interaction studies.
VibriClone
Our strain VibriClone is engineered to be a chassis organism for accelerated cloning.

Why do we actually use E. coli for cloning? What are the reasons why E. coli is the one and only workhorse for molecular cloning? A big milestone in molecular biology was the first cloning in the 70's with the organism E. coli. Since this eventful step, the cloning has been constantly improved and accelerated. In addition, the strains of E. coli used for the cloning were adapted as best as possible to the requirements.
But what about the selected Chassi? Of course, E. coli is a good choice because it is genetically simple, has a fast growth rate and is now well studied. However, there are so many different microorganisms, maybe there is a species that is even better suited for cloning. We introduce to you the organism, it is Vibrio natriegens. Imagine you have all the advantages of E. coli for cloning, but much faster and easier. Through the transition, it would be possible for us scientists to complete cloning in one day instead of three and as you all can imagine, that would speed up the entire science. The only thing missing so far to use V. natriegens as cloning host is the optimization and the characterization. Exactly this point is our project. We want to make V. natriegens accessible and useful for you so that you have a great advantage for your important work by the changeover.

Exonucleases and Endonucleases

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Figure 1:
The wildtype E. coli strain has same nucleases. They are divided into two types: exonucleases and endonucleases. Exonucleases play an important role in cells in the repair of DNA, genetic recombination and mutation avoidance. The enzyme hydrolyzes the phosphodiester bonds in DNA from a free end (Lovett, 2003) .Endonucleases are enzymes that cleaves hydrolytically the internal phosphodiester bond of DNA and/or RNA and this leads to their degradation. Bacterial endonucleases are also called restriction enzymes. In contrast to Exonucleases multiple nucleotides arise (Nishinoet al.2002) (Figure 1).

Vibrio cholerae, like V. natriegens, is able to pick up DNA from the environment. However, most of the DNA is degraded by the nucleases secreted by the microbial community. Some transformable material is mostly available and can be taken up by naturally competent cells. V. cholerae has two extracellular nucleases: xds and dns. It has been shown for V. cholerae that the transformation efficiency greatly increases when the two extracellular nucleases are knocked out. Thus, the DNA to be transformed into the organism is no longer degraded (Blokeschet al.2008) . In order to build a cloning strain of V. natriegens, it is also very important to knock out the nucleases. Otherwise, the DNAses would digest the DNA and it would not be possible to transform DNA.

RecA

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Figure 2:
The enzyme RecA is present in many bacteria. It is an ATPase that mediates homologous recombination. Homologous recombination is important for genetic diversity and genomic integrity (Chenet al.2008) . The role of the allosterically regulated protein in this process is to detect the homologous sequence in a variety of heterologous sequences (Saviret al.2010) . RecA - bound to ATP - can make a complex with single-stranded DNA. This helical filament can bind to double-stranded DNA, search homologies, and then catalyze the exchange of the homologous strand (Chenet al.2008) . In addition, recA has another function. It has a co-protease function in the bacterial SOS response system being responsible for cleavage of LexA and the λ-repressor (Horiiet al.1981) . Because you do not want to have a chromosome integration or homologous recombination in a Cloning strain, this gene is mutated in the E. coli strains. For our VibriClone we also want to have a greatly reduced single-stranded DNA-dependent ATPase activity for RecA (Bryan, 1988) (Figure 2).

KatG

The katG gene encodes an enzyme with a catalase and peroxidase activity. Peroxidases use hydrogen peroxide as a substrate. A special form is the Catalase, which is also KatG. Catalases are enzymes that catalyze the reaction of hydrogen peroxide to water and oxygen. The fact that they make hydrogen peroxide harmless has a protective effect on the cell. Many aerobic organisms including E. coli have a catalase because they contain flavoenzymes that contribute to peroxide formation (Fuchs 2017) . Due to the catalase activity it is possible to store E. coli at low temperatures, which is very useful in the laboratory. That's what we want for our VibriClone, too. Because we will have the working with VibriClone as comfortable and practical as possible.

The lac operon

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Figure 3:
The lac operon plays an important role in the transport and the degradation of lactose in bacteria and it is a section on the chromosome of E. coli. It contains the three genes lacZ, lacA and lacY, which are regulated together. They encode enzymes for the utilization of lactose. The lacI gene is no longer counted to the lactose operon. The Lactose repressor (LacI) has an central importance for the regulation of the expression of the lactose operon. This is an enzyme that occupies the cell membrane of the bacterial cell and is responsible for the transport of lactose into the cell (Santillánet al., 2008) . The ß-galactosidase (LacZ) convert lactose to glucose and galactose (Serebriiskiiet al., 2000) . The glucose can then be metabolized normally by glycolysis. The exact function of the Thiogalactoside transacetylase (LacA) is unknown, but it may act as an detoxifying enzyme. Transcription of the lactose Operon is regulated by both the repressor and the cAMP-CAP complex. The lac operon is negatively regulated by a repressor and positively by an activator. In addition, the activity of the lac permease is controlled by the inducer exclusion mechanism. The lactose operon works the following way. When lactose is present in the surrounding medium and there is no energy source more favorable for the cell like glucose for cellular metabolism, the three genes of the lactose operon are expressed. In the following lactose is transported through the permease into the cell and there is decomposed by the enzyme ß-galactosidase into glucose, galactose and allolactose. The allolactose performs a backbond to bind to the lactose repressor and allows the transcription to proceed. Negative and positive regulation controls the degradation of the most efficient source of energy (Figure 3).
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Figure 4:
If a strain has integrated the lactose operon, it is possible to perform a blue-white selection (Figure 4). The basic idea of the genetic engineering principle is to check whether the genetic modification was carried out correctly. The ß-galactosidase, the gene product of the LacZ genes, consists of two components: LacZ-alpha and LacZ-omega only if both components are functionally present, galactosidase can exert its catalytic effect (Langleyet al., 1975) . In the DH5𝛼 strain of E. coli the 𝛼 subunit is deleted. Now, a plasmid containing the alpha subunit is used. (Zamenhofet al., 1972) . By a successful transformation of the plasmid into the mutated strain, a funcional ß-galactosidase can be expressed. If an insertion into this vector occurs, the complementation can not work. After transformation, the cells are plated on selection medium. It contains IPTG, which makes the lac repressor inactive and allows expression of the lacZ gene. In addition, X-Gal is included, which is split by the ß-galactosidase in a blue product (Burn 2012) . This means that the white colonies have integrated the plasmid with insert and the blue colonies without insert. This simple method makes it quick and easy to see which are the right colonies that you can pick. Our big goal is to make this method usable for the VibriClone as well. This would give you a huge time advantage through the rapid growth of V. natriegens and the rapid recognition of the result. After many rounds of designing, and engineering Vibrio natriegens to a suitable clone strain, we now have VibriClone in our hands, with all properties for a perfect cloning host, such as nuclease deficience, recA mutation, cold resistance and the ability for blue white screening.
We want to make our strain accessible to the whole iGEM community by providing the opportunity to request our VibriClone strain, to enable all future iGEM Teams to accelerate their projects with the next generation cloning chassis.
VibriExpress
Our strain VibriExpress is engineered to be a chassis organism for accelerated heterologous protein expression.

Nowadays the production of high protein amounts is important for biotechnological and industrial uses. One of the widely used expression hosts is E. coli in combination with the T7 expression system. For the last century, the performance of E. coli was hugely satisfying, except of issues like the aggregation of inclusion bodies and the insolubility of some proteins (Villaverde & Carrio 2003). However, in the last years, scientific progress is getting faster and faster and it is time to question if there is an alternative to E. coli, a host that is keeping up with the time?
Imagine you could start a day early in the morning performing a transformation with your construct. After several hours, you could inoculate colonies in liquid culture and induce the expression of your desired protein in an overnight culture. The second day you would be able to purify your protein. Using V. natriegens as a new host with its impressive doubling time and the ability to grow to a much higher cell density will arise new possibilities for molecular and synthetic biology. In order to do so, we will design the VibriExpress strain.

“Creating a strain that is optimized for efficient production of high quality protein material is of high value for the drug discovery process.”
this was the view of Dr. Serghei Glinca, the Chief Executive Officer of CrystalsFirst, a company that is specialized on protein-crystal based applications in drug discovery. Click here to learn more about our Integrated Human Practices project. The functionality of the T7 system in V. natriegens was already demonstrated and additionally it is pointed, that V. natriegens yields higher amounts of soluble protein than E. coli (Weinstock, Hesek et al.,2016).
But how is the commonly used T7 system working?

The T7 system

The system is based on the bacteriophage T7 RNA Polymerase, that shows a very high activity and synthesizes RNA at a rate several times that of E. coli. Besides, it is highly selective for its own promoter. Combined with the ability to inhibit selectively the host RNA polymerase with rifampicin (Hartmann et al., 1967) , it allows the exclusive expression of genes under the control of a T7 RNA polymerase promoter (Tabor S., 1985) . Due to these advantages, the gene of the T7 RNA Polymerase was integrated into the E. coli BL21 genome and is controlled by a lacUV5 promoter (Moffatt et al, 1986) , (Jun Lee et al., 2015) . The gene of the desired protein is introduced into the cell using a vector with a T7 promoter (e.g. pET vector, commercialized by Novagen). Incubating the cells with IPTG leads to the expression of the T7 RNA Polymerase. Finally, the Polymerase recognizes the T7 promoter and the expression of the desired protein is enforced (Figure 1) (Steitz et al., 1999) . To maximize the produced protein amount, proteases like Lon or OmpT can be knocked-out (Dunn et al., 1988) .

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Figure 1:

The Lon protease

The Lon protease derives its name from the phenotype of E. coli lon gene mutants that form long undivided filaments upon UV irradiation (Simson et al. 1964) . It is a component of the protein quality-control systems and degrades misfolded and unfolded proteins in an ATP dependent manner (Chung & Goldberg 1981) and is also involved in the degradation of certain regulatory proteins like SulA, which is a component of the SOS system and an inhibitor of cell division (Mizusawa & Gottesman 1983) , Huisman, (D'Ari et al. 1984)).

The OmpT protease

The other deleted protease OmpT is an aspartyl protease localizing at the outer membrane of E. coli. It is suggested that OmpT is involved in the pathogenicity of E. coli. More than 30 years ago is was shown that OmpT catalyzes the activation of plasminogen to plasmin (Bowles et al., 1981) . This function seems to be physiologically relevant for the virulence of clinical E. coli isolates (Lundrigan & Webb 1992) and other pathogens like Yersinia pestis (Subrahmanyam et al., 1992) . Additionally the level of OmpT expression was shown to increase in response to the induction of recombinant protein overexpression (DeLisa et al., 2000) . For T7 expression strains, the deletion of this protease is urgently required because it was also shown that OmpT is degrading T7 RNA Polymerase (Grodberg & Dunn 1988) . According to NEB (New England Biolabs Ipswich, Massachusetts) strains that are deficient in both proteases Lon and OmpT are more amenable for the production of proteins

Our vision is to create a strain similar to E. coli BL21 and maximize the protein yield to an optimum by not only integrating the T7 system into V. natriegens but also deleting proteases to decrease protein degradation during the purification process. This way we will design an optimized strain for high-speed and high-level protein expression. Making VibriExpress accessible for everyone, we would like to accelerate your research and support you all, making the world a better place.

VibriInteract
Our strain VibriInteract is engineered to be a chassis organism for accelerated protein interaction studies based on the Bacterial Two Hybrid system.

Proteins are part of the building blocks of life and fulfill a variety of essential functions. But what could one protein alone achieve? Mostly proteins interact with partners or in complexes to fulfill their function. A general goal of science is to understand all biochemical processes that enable life as we know it. Thus, the understanding of protein interaction partners becomes more and more important.
It is crucial to have methods investigating interactions between different proteins since most proteins do not act alone but in complexes with other proteins. This circumstance is of central interest since it can reveal a lot about the function of a protein and the processes that it is involved in. Using those methods, one can study interactions of specific proteins but it is also possible to screen for potential interaction partners using a library of genes. The latter one was already used by the iGEM team Bielefeld-CeBiTec in 2016. In their project they established their own method to do interaction assays to screen for proteins with an innate affinity for a specific target (Click here to learn more about the Bacterial-Two-Hybrid assay developed by iGEM Bielefeld-CeBiTec). This shows that interaction studies can not only be used in basic research but could also play an important role as an application in synthetic biology.

Protein interaction studies can be done by several methods:

  • Biochemical Methods
    • e.g. Co-immunoprecipitation
  • Biophysical and Theoretical Methods
    • e.g. Fluorescence Resonance Energy Transfer (FRET)
  • Genetic Methods
    • e.g. Two-Hybrid Systems

A lot of these methods still need a lot of time to be carried out. This can be done faster! Therefore we created our third strain: VibriInteract. A strain which can be used for protein interaction studies.

For Two-Hybrid Systems, the chassis organisms Escherichia coli and Saccharomyces cerevisiae are used in a Bacterial Two-Hybrid (B2H) and Yeast Two-Hybrid (Y2H) system, respectively. The Y2H assay was a novel genetic system at that time used to detect protein-protein interactions heterologously in yeast (Fields & Song, 1989). 9 years later, a new system was proposed. Instead of S. cerivisiae, this system uses E. coli as chassis organisms (Karimova et al., 1998). That this genetic test is carried out in E. coli greatly facilitates the screening as well as the characterization of the interacting proteins. Moreover, it accelerated this assay since E. coli has a much faster growth than S. cerivisiae. Although B2H assays are the easiest and fastest methods to test for interactions nowadays we think that they can be done faster. Using E. coli (which is already faster than using an Y2H system), the assay still needs 24 to 72 hours of incubation. Using V. natriegens, we can accelerate this assay to 8 to 24 hours of incubation. Paired with one day cloning we can speed up this assay from 7-9 days if your work in E. coli to 3 days if you work in V. natriegens. By this, we create our very own Two-Hybrid Assay: Vibrio Two-Hybrid (V2H).

An ordinary workflow of a Bacterial Two Hybrid Assay An ordinary workflow of a Vibrio Two Hybrid Assay
3 days cloning (at best)
2 - 4 days first transformation
2 - 4 days spot assay
1 day of cloning (at best)
1 day first transformation
1 day spot assay
7-9 days in total 3 days in total

The Bacterial Two-Hybrid system is based on reconstitution of a signal transduction pathway relying on the adenylate cyclase (CyaA). CyaA is a key regulator in a lot of cells like e.g. E. coli. It catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) (Figure 2 A) (Krupinski, 1991). cAMP binds to the catalytic activator protein (CAP). This interaction effects a conformational change which leads to a higher binding affinity to DNA. The complex binds to the CAP site upstream of the promoter and can interact with the RNA polymerase (Kolb et al., 1993). This interaction increases the affinity of the polymerase to the promoter. The positive regulation boosts the expression of the gene by a factor of approximately 50 compared to gene expression without the presence of cAMP (Figure 1). To conclude, the cAMP/CAP complex is a positive regulator of e.g. the mal operon and the lac operon.

Schematic figure of how cAMP is involved in regulation of gene expression.
Figure 1: Schematic figure of how cAMP is involved in regulation of gene expression.

For B2H an E. coli strain lacking the cyA gene, which is encoding the catalytic domain of the adenylate cyclase and does not produce cAMP, is used. Consequently, cells can no longer utilize maltose or lactose. This phenotype can be rescued using the complementary T25 and T18 fragments of the catalytic domain of the adenylate cyclase toxin from Bordetella pertussis. Those fragments are cloned to genes of interest in plasmids called pKT25 and pUT18, respectively. Those fragments when splitted can no longer synthesize cAMP (Figure 2 B). If two hybrid proteins interact in this assay the two fragments of the catalytic domain come into close proximity and produce cAMP(Figure 2 C). The cAMP/CAP complex is formed again and regulates the mal or lac operon positively. Cells containing interacting constructs can utilize maltose and lactose again. Based on this, interaction of proteins can be screened using methods based on the lac operon or the maltose operon. For this, several different media are used:

  1. LBv2 agar plates supplemented with IPTG and Xgal used for Blue-White Screening if the lac operon is functional. You can read more about Blue-White Screening in the VibriClone part of our description.
  2. MacConkey agar plates supplemented with maltose to test if the mal operon is functional. When cells utilize maltose, acids are formed and cells turn red because of the pH indicator neutral red.
  3. M63 minimal medium agar plates supplemented with maltose to test if the mal operon is functional by screening for growth of colonies.

Principle of a Two-Hybrid system based on reconsitution of the adenylate cyclase.
Figure 2: Principle of a Two-Hybrid system based on reconsitution of the adenylate cyclase of B. pertussis. (A) The full-length catalytic domain can synthesize cAMP. (B) T25 and T18 fragments, when coexpressed as splitted peptides, are not able to interact and cAMP is not synthesized. (C) Both fragments, when fused to interacting proteins, get into close proximity and cAMP is synthesized.

CRISPR/Cas9
In the last few years, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) method became a popular application in genetic engineering. This method is based on an adaptive antiviral defense mechanism of prokaryotes (Barrangou et al. 2007). As a part of the bacterial immune system, CRISPR/Cas provides protection against foreign DNA through the action of the RNA-guided DNA endonuclease Cas, which cleaves the entering viral DNA at a specific point (Qi et al. 2013).

The first CRISPR-loci were discovered in 1987 in the E. coli strain K12  (Ishino et al. 1987) and the first description of a cas gene and their homologies to DNA-related enzymes appeared in 2002 (Jansen et al. 2002). Since then, more than 9000 research articles have been published about the different CRISPR/Cas subtypes and their application in various CRISPR based technologies (Adli 2018). The potential applications are almost limitless, ranging from new methods in research to industrial applications as well as human gene therapy. With CRISPR/Cas, it is possible to specifically switch off genes in a large number of prokaryotic and eukaryotic organisms, to integrate foreign DNA into the genome of an organism or to use the DNA-binding property for in vitro experiments (Hsu et al. 2014).

Figure 1: CRISPR/Cas9.
The picture shows the Cas9 assembled with the sgRNA and DNA. The scaffold of the sgRNA is illustrated in purple and the spacer in blue. The PAM is shown in red.
Due to its simplicity and modularity, the Type II CRISPR/Cas9 system from Streptococcus pyogenes is a commonly used tool for genetic engineering applications in a variety of organism (Hsu et al. 2014, Charpentier & Marraffini 2014, Doudna & Charpentier 2014). For its RNA-guided silencing of foreign DNA it requires just one Cas9 encoding gene and the action of the CRISPR RNA (crRNA) as well as the trans-activating crRNA (tracrRNA) (Jinek et al. 2012, Gasiunas et al. 2012). During infection, the CRISPR-locus will be transcribed into the pre-crRNA containing the spacer region flanked by conservated repeats. The tracrRNA binds to the pre-crRNA due to 25 nucleotides which are complementary to the repeat regions of the pre-crRNA (Deltcheva et al. 2011). After procession, the mature crRNA:tracrRNA duplex binds the Cas9 with the tracrRNA builded stem-loop constituting a CRISPR ribonucleoprotein (crRNP). The crRNA contained a 20 nucleotides large spacer region at the end of the N-terminus which is complementary to the invading DNA (protospacer) therefore leading the crRNP to the target. In addition, the presence of the Protospacer adjacent Motif (PAM, NGG in S. pyogenes) preceding the protospacer three nucleotides is necessary for the cleavage activity (Jinek et al. 2014, Nishimasu et al. 2014). The crRNA and tracrRNA can directly be linked by a linker loop into a single guided RNA (sgRNA) like we used in our project for a more simplified and modularized genetic engineering application (Brazelton et al. 2016, Jinek et al. 2012).

Metabolic Engineering
We want to show the suitability of V. natriegens for Metabolic Engineering due to its genetic accessability, fast growth and high substrate-uptake rates.
Our vision of Accelerating Metabolic Engineering

Our Goal

Our goal is to accelerate metabolic engineering by establishing a workflow for fast pathway construction, product screening and pathway optimization. Metabolic engineering is a broad field in synthetic biology, where existing pathways are divided into modular parts and then used and combined to build new pathways (Stephanopoulos et al.2012) . One of the best examples for the impact of metabolic engineering on solving global problems is the engineered production pathway for artemisinic acid, the precursor for a drug against malaria (Ro et al.2006) . In the old days the drug had to be isolated out of the plant Artemisia annua. This was very inefficient because of the limitations of plant growth and low yields of artemisinic acid. Therefore, the group of Jay Keasling engineered a metabolic pathway to produce the drug in yeast cells, which grow faster and produce a lot more artemisinic acid. As in this example, metabolic engineering enables the production of drugs and other valuable products with an increased yield when compared to product isolation from natural sources. Furthermore, the large variety of usable enzymes, pathways and chassis enables more flexibility in pathway design compared to chemical approaches.

Why should we accelerate metabolic engineering?

Although much progress in metabolic engineering was made in the last 20 years, there are still many drawbacks and limitations. For instance, it is crucial to have a big set of genes as combinable parts to find the optimal pathway and they have to be predictable. With many of them just poorly characterized, rational pathway design remains difficult. Our understanding of the working and interactions of enzymes at the level of the metabolism is still rudimentary. Consequently, metabolic engineering consists of much trial-and-error and researchers have to endure series of failures. One strategy to face these issues is modeling of enzyme behavior but currently limitations in computing power prevent this from being used in high-throughput. Another strategy is to use chassis as workhorses to test a lot of enzyme combinations and pathway variants. Here, organisms like E. coli, S. cerevisiae or C. glutamicum are often used but product formation is limited by growth rate and nutrient uptake (Stephanopoulos et al.2007). When a pathway is constructed and to be tested, one has to wait until the production strain has grown and then product formation is still limited by substrate uptake. Another big drawback is that screening for product often is expensive, time consuming and complicated. Conventional methods like LC-MS need a lot of time and resources and it is hardly possible to use it in high-throughput, for instance for a whole pathway library. To overcome these limitations and accelerate metabolic engineering, we established V. natriegens as chassis for metabolic engineering. Its doubling time of under 10 minutes makes it the perfect organism to quickly test many pathway variants or enzyme versions. Additionally, it has a glucose uptake rate of 3.90 g g-1h-1 while E. coli, S. cerevisiae and C. glutamicum have much lower rates (1.90, 3.52 and 0.37 g g-1 h-1, respectively). Coupled with the short doubling time, V. natriegens is a powerful chassis not only for research purposes but also for industrial usage, where high productivity is crucial.

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Figure 1: Metabolic Workflow established by iGEM Team Marburg 2018

How are we accelerating metabolic engineering?

For optimal execution of metabolic engineering we established a workflow, basing on the DBTL-cycle. We use our Marburg Collection to build whole pathway libraries, which can then be transformed into our producer strain VibriXpress. By using biosensors for product screening, we circumvent the expensive and time-intensive usage of LC-MS and learn which pathway variants are the most promising. These findings can then be fed into our pathway-design, thereby closing the DBTL-cycle. To further optimize these pathways, we planned to use directed evolution for more adaptations to that route.

Production of 3-hydroxypropionic acid

3-Hydroxypropionic acid 3HPA

As a proof of concept for our engineering workflow we chose the biological production of 3-Hydroxypropionic acid ( 3HPA ). 3HPA is a compound of high industrial value. In 2004 the U.S. Department of Energy recommended 3HPA as alternative to fossil oils for the chemical industry (Werpy et al.2004). 3HPA as a platform chemical can be converted into many other compounds like acrylic acid and acrylamide, which both are also precursors for further compounds. According to an estimate of 2014, acrylates alone have an annual market value of USD12 billion, making its precursor 3HPA an optimal target product for our metabolic engineering approach. By finding the optimal pathway to produce 3HPA we could show the efficiency of metabolic engineering in V. natriegens as well as getting a big step closer to an alternative to fossil oils by biological, renewable resources.

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Figure 2: Industrial applications of 3-hydroxypropionate.

Our pathway

In recent years many people concentrated on producing 3HPA via many different metabolic pathways (Valdehuesa et al.2013) ; (Vidra et al.2017). Most of these pathways use glycerol or glucose as starting substrate, but there are also publications where acrylic acid, CO2 or uracil were converted to 3HPA (Vidra et al.2017). One promising route starts with glycerol which is dehydrated and oxidized. For E. coli, a productivity of 6.6 mmol g-1 cdw h-1 (oder Lieber andere units? 6.5mmol l-1 oder 0.48 mol mol-1 glycerol) has been shown (Raj et al.2008). However, this route depends on vitamin B12, which often can’t be taken up or produced by the chassis. Many other routes start with glucose, which gets metabolized via propionate, lactate, β-alanine or malonyl-CoA (Vidra et al.2017). We decided to use a pathway, based on the conversion of acetyl-CoA into malonyl-CoA and finally 3HPA . According to (Valdehuesa et al.2013) who evaluated many pyruvate-derived production pathways from a thermodynamic point of view, this is one of the most efficient routes for 3HPA production. This pathway was first established in E. coli, but it was also tried in many other chassis like S. cerevisiae and S. elongatus (Kildegaard et al.2016); (Liu et al.2017); (Rathnasingh et al.2012). We chose this route for several reasons. Firstly, it is based on glucose degradation and V. natriegens has an unbeaten glucose-uptake rate, enabling high glucose consumption. Secondly, vitamin B12 is not necessary, the only cofactors involved are NADPH and biotin, both of which occur naturally in the organism. Thirdly, theoretically just one further enzyme is needed to complete the pathway. All enzymes necessary for the production of malonyl-CoA are already present in V. natriegens and we just have to integrate the last enzyme, malonyl-CoA reductase. Nevertheless, overexpression of acetyl-CoA carboxylase increases production, so we took both enzymes into consideration. (Liu et al.2017) proposed, that there are many ways to further optimize the pathway and direct the flow towards a high product titer. Hence, implementing many variations of this pathway into V. natriegens and testing them in a fast manner promises a wealth of knowledge about pathway optimization and general principles of metabolic engineering.

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Figure 2: Pathway we have chosen for our metabolic engineering efforts.
The first relevant enzyme in the pathway is the acetyl-CoA carboxylase (Acc) that catalyzes the carboxylation of acetyl-CoA. It is involved in fatty acid biosynthesis,consisting of four subunits in E. coli, a biotin-carboxylase (BC), a biotin-carboxyl-carrier-protein domain (BCCP) and two carboxyltransferase subunits (CT) (Jansen et al.2004). During catalysis, a biotin molecule is first linked to a lysine residue in the BCCP domain. Via ATP-hydrolysis the BC domain carboxylates the biotin, which then induces a conformational change and brings the carboxy-group into close proximity of the CT domain. Finally, the CT domain transfers the carboxy-group to the acetyl-CoA, leading to the formation of malonyl-CoA (Lee et al.2008) The subunits are organized in four genes in E. coli (Rathnasingh et al.2012) , however, in other organisms like C. glutamicum the genes are located in two operons (Gande et al.2007).
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Figure 3: Mechanism of ACC
The second relevant enzyme in the pathway is the malonyl-CoA reductase (Mcr), which is involved in CO2 fixation by the 3-hydroxypropionate bicycle in thermophilic bacteria (Alber et al.2002). It is a 132kDa large, bifunctional enzyme with alcohol dehydrogenase and aldehyde dehydrogenase function. The C-terminal domain catalyzes the NADPH-dependent reduction of malonyl-CoA into 3-oxopropanoic acid, while the N-terminal domain catalyzes consecutive reduction of 3-oxopropanoic acid to 3-hydroxypropionate (Liu et al.2013).

Sensor-based product screening

One major goal of synthetic biology is to make biology engineerable and predictable. Currently metabolic engineering does still involve a lot of trial and error. With the number of possibilities for pathway finetuning this scales very fast, becoming very difficult to tackle with traditional methods.

Also, once we create such a library of metabolic pathway variants, possibly including thousands of strains, we have to find the most promising ones among these. A traditional approach would be to check the production of each strain in its own culture flask. Using for example HPLC or mass spectrometry.

A more suitable, easy way to quickly screen for metabolite concentration in a semi quantitative way are biosensors. One of the easiest to use classes of biosensors are metabolite-responsive transcription factors (Liu et al. 2018). They are usually very specific towards their distinct substrates. In vivo, most of these systems help their native organism to react to environmental clues by regulating expression of for instance catabolic enzymes or importers.
In the past, these have ben co-opted by researchers to function as sensors or to control enzyme expression to regulate a pathway depending on the availability of a certain metabolite (Rogers, Taylor, and Church 2016). By using reporter genes like GFP or the LUX operon, we have a measurable output that can be used to select for the best contenders. By running this in several iterations, nature's most powerful optimization tool, evolution, can be applied to further improve the pathway (Williams et al. 2016). Applying fluorescence activated cell sorting (FACS) coupled with V. natriegens incredible doubling time, thousands of generations can be screened for progressively better pathway variants.

So, we decided to put our toolbox full of characterized parts to good use. The Marburg collection contains all the parts needed to find the ideal enzyme balance for highest production. Being able to build a large number of differentially assembled pathways is the answer to creating the most productive strain. Finding the right one in that complexity is another difficulty to overcome, one we solved by finding transcriptional regulators that specifically activate reporter gene expression relative to the concentration of key metabolites, 3-hydroxypropionate and Malonyl-CoA. This gives us a quantifiable output with which we are able to judge the different combinations. Also, utilizing the speed of V. natriegens , the time required for a high throughput screening can be reduced dramatically.

We deemed the concentration of two different metabolites as being critical to our project: 3-Hydroxypropionate, the product itself, and malonyl-CoA, an important intermediate we also plan to overexpress.

This is a schematic depiction of the mechanism for the 3-HPA sensor. The constitutively expressed HdpR protein can bind 3-HPA. When this happens, a conformational change occurs. The changed enzyme can bind to the activator region and drive reporter gene expression
Figure 1: Scheme of the working mechanism for the 3-HPA sensor

Using the information from the paper of Hanko et al. (2017), we settled for the hdpR / hdpH system coming from Pseudomonas putida KT2440. It consists the constitutively expressed HdpR protein (BBa_K2560304). , which is able to bind to the P HdpR and induce expression of a gene. In the native setting, that would be the hdpH gene, encoding for a 3-hydroxypropionate dehydrogenase which enables P. putida to grow on 3HPA . We cloned our reporter gene in its position. This way we got a direct readout of the 3HPA concentrations in the individual cells.


Because our model showed that the low activity of V. natriegens native Acc , malonyl-CoA is likely to be a limiting factor in 3HPA production. Having a direct indication to how much malonyl-CoA is available

figure showing the working mechanism of the malony-coa sensor. The fapR protein is expressed constitutively. when it binds 3hpa, a conformational change occurs, enabling it to bind the fap0 represor binding site. This binding site is so close to the promoter controlling our reporter gene, that once fapR has bound to fapO, expression of the reporter is inhibited.
Figure 2: Scheme of the working mechanism for the malonyl-CoA sensor
and how our efforts to introduce heterologous Acc´s succeed in elevating the native levels would be quite helpful. This can be achieved in a quite similar way to the 3HPA measurement. Our literature research quickly gave us several starting points. One of them, a system from Bacillus subtilis , in which the repressor FapR binds the 17bp motif fapO, thereby sterically hindering polymerase binding to an adjacent promoter(Liu et al. 2015). When the FapR protein binds malonyl-CoA, a conformational change is triggered which results in FapR dissociating from the fapO site. The now accessible promoter drives expression of the gene under its control, which we replaced with a reporter.

Directed Evolution and Applications

Directed evolution

Although the Marburg Collection enables the construction of many pathway variations, there are still many more possibilities for direct metabolic fluxes towards 3HPA . To enforce these flux improvements, we planned to use directed evolution coupled with FACS selection. By implementing genes for the conversion of 3HPA into succinate, we lead our product back to the central metabolism. By deleting all other genes responsible for succinate production, the cells are forced to use the 3HPA bypass to get succinate, which is needed in the TCA cycle. The big advantage of using V. natriegens for that purpose is that it’s short doubling time increases the number of cell divisions per time and thereby increases the number of mutations in a given time frame. Coupled with our biosensors and fluorescence-activated cell sorting (FACS) we can easily and rapidly select for cells performing the most efficient pathways. Additionally to mutation-guided evolution there are many genome modifications which could increase flow towards 3HPA , for instance by downregulation of side reactions. In the Design-Section a list of possible genome modifications is shown. [Link zu Directed evolution im Designabschnitt machen]. By using MAGE , it is possible to get a library of different genome modifications, which can then be selected via FACS for their efficiency to produce 3HPA or succinate. MAGE stands for multiplex automated genome engineering and is a technique to create combinatorial genome modifications. MAGE uses a set of oligos, which get transformed into a population of cells and then integrate into the chromosome via homologous recombination. Different cells in the population will have different numbers and combinations of modifications leading to a genomic diversity. By applying MAGE in combination with FACS for our pathway we get a huge variety of possible pathway optimizations which can be sorted via FACS based on their efficiency.

Applications of our workflow

By using V. natriegens as a chassis for metabolic engineering, it is possible to build, test and optimize pathways and enzymes at a high-throughput level. The Marburg Collection enables the fast construction of pathway libraries, while the genetic accessibility of the strain makes genome modification and pathway integration simple. The usage of our biosensors for screening of malonyl-CoA and 3HPA accelerates identification of the best pathway variants and the short doubling time coupled with the high nutrient uptake rate empowers V. natriegens to be the perfect chassis for metabolic and enzyme engineering!

Vibrio natriegens, a review
Science is a collaborative effort, we all build on the work of others. Sometimes, finding all relevant sources can be difficult, so we wrote a short review of the research into V. natriegens from the last 60 years.

When we first started research about V. natriegens, we only came across the latest publications, as well as the original descriptions. Only very tentatively, we became aware of the full body of research that had accumulated since its discovery. In order to make it easier for the scientific community to become familiar with V. natriegens, we thoroughly collected all available publications about it. You can find the complete list of publications on our wiki. The following text is meant to give a first impression about what is known and what has been done so far regarding V. natriegens.

Introduction

First isolated from salt marsh mud on Sapelo island off the coast of Georgia in 1958 (Payne et al. 1958), the gram-negative bacterium was first named Pseudomonas natriegens (Payne et al. 1960). These early studies also revealed a broad range of tolerated pH conditions with an optimum at a pH of 7.5 (Payne et al. 1960). One feature of this newly discovered organism became immediately apparent: The incredible doubling rate, first determined to be around 9.8 minutes (Eagon 1962). We found that doubling rates of around 7 minutes were possible (own Data).

Only much later, studies found several possible reasons for this. Firstly, as most Vibrio species, V. natriegens genome is split and distributed on two chromosomes. This means that replication can start at two origins of replication (Ori), resulting in more in parallel replication.

picture showing V. natriegens with long, polar flagella
Figure 1: EM photograph of V. natriegens (Austin et al. 1978).

Additionally, it was shown that the gamma-proteobacterium has an increased rRNA activity. This is because of a greater number of rRNA operons which are additionally controlled by stronger promoters (Aiyar et al. 2002) when compared to E. coli . More rRNA means more ribosomes since rRNA synthesis has been shown to be the rate-limiting step in their assembly (Miura et al. 1981). Estimates for the number of ribosomes in V. natriegens in the exponential phase suggest around 115,000 per cell, while E. coli is estimated to have between 70,000 and 90,000 (Failmezger et al. 2018).

This gives V. natriegens higher protein expression and lets it create biomass more quickly. Could this also be exploited for the production of high-value proteins? Most likely.

It was soon discovered that V. natriegens would make a perfect example by which to effectively and harmlessly demonstrate the basic techniques of microbiology to students, taking full advantage of the immense doubling rate (Mullenger 1973; Delpech 2001). Experiments regarding population structure, UV stress, and simply bare growth can be observed in a much shorter time frame.

V. natriegens has a single stage lifecycle and does not form spores. The rod shaped bacterium does however possess a single polar flagella (Austin et al. 1978).

Ecology
V. natriegens has some amazing abilities. By understanding them as adaptations, it becomes quite apparent that V. natriegens is itself first and foremost a product of its environment. Therefore, getting a better understanding of its ecology helps explain some of these capabilities.
picture showing a salt march like the one V. natriegens wass first isolated from
Figure 1: Coastal salt marsh in the southeast USA, this is the habitat V. natriegens was first isolated from(Pendragon 1966).

The rod-shaped bacterium occupies a special ecological nice in estuarine and salt marsh regions, where strongly fluctuating salt concentrations and nutrient availability create a challenging environment. In order to adapt to these challenges, V. natriegens can utilize a wide range of substrates to outgrow competitors, as well as remain in a low metabolic state for extended periods of time when the nutrient pool is depleted (Nazly 1980). Furthermore, it has the ability to quickly draw available phosphorous from its environs and store them in polyphosphate bodies (Nissen et al. 1987) for later utilization. Yet another way in which V. natriegens is adapted to its variable environment is the storage of carbon as polyhydroxyalkanoates (PHAs), biodegradable polyesters that are accumulated in intracellular granules that can be broken down under starvation conditions (Chien et al. 2007).

From the well oxygenated upper layer of mud down to the anoxic layers, V. natriegens is able to grow in many surroundings. These extremes are challenges to which V. natriegens found solutions. At the surface of the mud, as well as in open waters, the bacteria are exposed to strong UV radiation. As a response to this, the DNA damage repair systems are highly active (Simons et al. 2010), with significantly elevated expression levels of some parts of the system compared to E. coli , thus increasing DNA sequence integrity. By switching to fermentation, growth can be maintained under anaerobic conditions, allowing the colonization of the lower strata in the mud.

V. natriegens was shown to be a vital part of the marine ecosystem because it has the rare ability to fix atmospheric nitrogen under anaerobic conditions, thereby enriching its habitat and playing a crucial role as a provider of essential, bioavailable nitrogen (Coyer et al. 1996) under anaerobic conditions.
picture showing several different phages isolated from V. natriegens cultures
Figure 2: Diverse phages isolated from V. natriegens cultures (Zacherey et al. 1974)

Unsurprisingly, V. natriegens has its own predators in its native habitat: Bacteriophages (Zachary 1974). Studies in the 70ies found a link between different phages and the prevailing environmental conditions (Zachary 1978) . This suggests the phages employ a divide and conquer strategy, specializing, thereby limiting competition while improving reproductive success. One phage may have adapted to be effective at lower temperatures, while others prefer higher temperatures. The same applies to other conditions like for instance salt concentration (Zachary 1976). Intriguingly, that opens the possibility of creating genetic parts derived from these phages, applicable to future work with V. natriegens, analogous to the T7 system in E. coli. On the other hand, one study could show, that the phage carrying the toxins associated with V. cholerae could not replicate in V. natriegens (Lee et al. 2016), further showing its harmlessness towards humans.

In contrast to that, some strains of Vibrio natriegens seem to be predators themselves. Confirmed as being pathogenic in several marine crustacea, most notably in the swimming crab Portunus trituberculatus, in which V. natriegens can lead to mortalities up to 85% (Bi et al. 2016). This is especially problematic since P. trituberculatus is farmed commercially in aquaculture in south-east China. The mechanism of that infection is poorly studied and it is not clear whether all strains of V. natriegens are pathogenic to crustaceans, but it is well known that many Vibrio species cause opportunistic infections in crustacea.

On a more positive note, a recent study could show that the sponge Aplysilla rosea develops a more diverse microbiome when exposed to V. natriegens (Mehbub et al. 2018). It can be speculated that this helps to protect the sponge from pathogenic microbes that then find it harder to colonize its surface.

Metabolism

A diverse, well-balanced metabolism allows a bacterium to grow in many conditions, while simultaneously making it interesting for biotechnological applications. V. natriegens can utilize a wide range of substrates (Hoffart et al. 2017), as well as grow in the absence of oxygen while being faster than other bacteria under the same conditions.

For many applications in which a modern laboratory work horse is expected to be useful, a high-resolution map of its metabolism is a prerequisite. One recent study generated just that (Long et al. 2017). By tracking 13C labeled carbon through the intricate network that constitutes V. natriegens metabolism, they were able to gain insight. Fore one, they found the it to be very similar to the well-studied E. coli metabolism. It has at the major catabolic and anabolic pathways, the same canonical amino acids and building blocks. Some notable differences between the two were the RNA content being 29% in V. natriegens compared to only 21% in E. coli, the presence of an enzyme for the decarboxylation of oxalate (Long et al 2017).

The specific uptake of many carbon sources per gram dry weight per time was sown to be significantly higher than in other comparable species of bacteria (Hoffart et al. 2017). Many commercially interesting compounds are already produced by unmodified V. natriegens. In the case of alanine production, only four deletions resulted in a strain that outperformed highly specialized E. coli and C. glutamicum production-strains by a factor of 9 to 13 times (Hoffart et al. 2017). Bioreactor based production on a large scale is expensive, therefore a strain generating similar or even higher yield per gram carbon source is highly desirable, improving temporal yield per fermentation unit.

When growing anaerobically, acids are produced that lead to an acidification of the medium but when grown under strong aeration, the pH can, depending on the medium, rise. This is most likely due to formation of ammonia (Eagon 1962).

The dependence on Na+ ions for its metabolic activity conveys a form of natural biocontainment, inhibiting growth in accidentally released V. natriegens (Webb and Payne 1971). The high salt content of the LBv2 media also presents possible contaminants with a barrier, reducing the likelihood of airborne microbes to take hold.

EM picture showing accumulated selenium nanobodies
Figure 2: EM picture showing accumulated selenium nanobodies in V. natriegens (Fern`ndez-Llamosas et al. 2017).

Recently, a study revealed that V. natriegens, surprisingly, is the most effective producer of selenium nanoparticles yet described (Fern`ndez-Llamosas et al. 2017). These particles with a diameter from 100-400nm have applications in diverse fields, such as medicine, microelectronics and more. Producing the nanoparticles in living systems has benefits over other methods, for instance, the low energy consumption as well as a coat of proteins preventing agglomeration of the particles. Also, V. natriegens exceptionally high resistance to selenite suggests possible applications in bioremediation of contaminated water and soils.

Working with V. natriegens

ATCC 14048 is the most commonly used strain of V. natriegens. Most results from earlier studies were generated from this strain. We recommend using ATCC 14048 in order to ensure that the findings will be applicable to your work and to adopt it as the standard strain. It is also the fastest strain known (Weinstock et al. 2016) so you won´t miss out on its incredible doubling time.

Stemming from subtropical regions, V. natriegens has not the same tolerance to low temperatures that we are used to from E. coli, but it is still much more resilient to cold than B. subtilis. At the root of that lies the low catalytic activity of V. natriegens native catalase at low temperatures, which is then unable to detoxify reactive oxygen species. To address that problem, scientists from SGI introduced homologous catalases, thereby creating a strain with comparable cold tolerance. With this adaptation, storage on plates can now be extended beyond the four weeks at room temperature by placing the plates in the fridge (Weinstock et al. 2016) In storage at -80°C in a standard 20% glycerol stock the cells stay viable almost indefinitely. And even when working with the wildtype, your cells won´t die overnight when placed at 4°C. Only after a week they will notably start decreasing in viability.

When working with V. natriegens ATCC 14048, no special precautions have to be taken since it has been shown that no known pathogenicity associated genes were present (Weinstock et al. 2016) Also, there is continued record of research since 1958 with not a single documented incident of a human infection with V. natriegens.

Molecular Biology
Accessing, altering and understanding the genetics of an organism has become commonplace since sequencing and methods for genetic engineering have become affordable and readily available. Making these tools available for V. natriegens is, therefore, paramount making it an attractive chassis.

Lately, a new branch of microbiologists developed an interest in V. natriegens. Their main focus lies in establishing state of the art methods and bringing V. natriegens to its full potential. We see our project as part of this movement and wish to make V. natriegens easily accessible to researchers. One groundbreaking paper (Weinstock et al. 2016) established several methods to make V. natriegens genetically accessible, as well as characterizing some central genetic parts. This paved the way for more research into applying its potential.

If you want to introduce genetic material into V. natriegens, you have several tried and tested options to choose from. You could use electroporation, a quick and simple method and the one with the highest transformation efficiency. Electroporation can also be used on wildtype cells. Or you could use chemically competent cells, the transformation works very similar to E. coli heat shock transformation. But in order to achieve good efficiencies using chemically competent cells, a nuclease deletion strain is needed. And if you want to insert especially large constructs, you can also use conjugation with very good success ( (Lee et al. 2016; Weinstock et al. 2016).

A fully sequenced genome is also a prerequisite for many studies. Currently, several sequences are available (Lee et al. 2016; Maida et al. 2013), one of them generated by us. The genome has a size of approximately 5.17 Mb. Automated annotation was performed, revealing more of the genomic structure, as well as enabling the reconstruction of the codon usage profile (Lee et al. 2016).

The genome of V. natriegens is, like in many species of the genus, split into two chromosomes. Chromosome one contains 3.24 Mb, chromosome two 1.92 Mb.

Plasmids, Promoter, resistance

Usually, a big hurdle when changing your chassis organism is a very basic question: Will my constructs still work? Do I need different ori? Do I have to redesign everything from scratch? Fortunately, the most commonly used origins of replication, ColE1, p15A and pMB1 are maintained just fine in V. natriegens (Weinstock et al. 2016) We could observe some differences in copy number, which we quantified. You can find the corresponding data on our Part collection page. To retrieve these plasmids from the cells, we have tested a wide variety of commercially available kits and found that all of them worked, returning plasmid of the same purity and yield as for E. coli. Promoter commonly used in E. coli also lead to expression in V. natriegens. Expression levels are slightly different, but we also quantified these and you can find the results on our toolbox page. The same goes for other basic genomic parts like terminators and ribosomal binding sites.

Commonly, when very high gene expression is needed, for instance in protein overproduction, the phage derived T7 system is used. The T7 phage infects E. coli but it was also successfully inserted into V. natriegens and shown to drive protein expression (Weinstock et al. 2016) One way of generating even stronger, phage derived promoters could be using V. natriegens derived phage expression systems.

Every organism has its unique set of resistances to certain antimicrobial molecules. In order to successfully use a new organism, awareness of its individual tolerances is required to use antibiotics successfully for selection. We could show that chloramphenicol, carbenicillin, tetracycline and kanamycin worked, but at different concentrations from E. coli. We measured the V. natriegens specific concentrations and made them available on our strain engineering page.

Plasmids in absence of a selective pressure were only retained for a short time (Own Data). Curing of plasmids that have no longer a purpose is therefore quick and fairly effortless.

MuGENT - Recombineering

Investigations into alternative genetic tools were also undertaken, most notably MuGENT and Recombineering.

MuGENT takes advantage of V. natriegens natural competence by inducing the TfoX system. The TfoX system, which is known from many Vibrio species, enables uptake and genomic integration of DNA parts via homologous recombination (Pollack-Berti 2010). In most cases, growth on chitin was identified as the trigger which activated the system. Regrettably, the trigger is not known for V. natriegens. Using this system, a group of researchers characterized this system in V. natriegens, making it usable by placing it under the control of an inducible promoter. Additionally, they demonstrated its potential by increasing the natural poly-hydroxybutyrate (PHB) production 100-fold (Dalia et al. 2017).

Recombineering takes advantage of the fact that short DNA oligonucleotides, that can easily be delivered into cells by a plethora of ways, can be used to generate point mutations, knockouts and small modifications in the genome, as well as on plasmids. The homologous flanks required are relatively short, allowing for constructs under 100 bp. Having this system available in V. natriegens means that generating new strains and gene variants is easy and quickly implemented (Lee et al. 2017).

Cell Free Protein-expression Systems (CFPS)

Very recently, several groups have published their work about cell free protein expression systems (CFPS) in V. natriegens (Failmezger et al. 2018; Des Soye et al. 2018; Wiegand et al. 2018). Cell free systems offer the advantage of direct access to many cell components as well as fine control over the conditions in the reaction (Carlson et al. 2012). While these are true for all CFPSs, the advantage of creating such a system from V. natriegens is the much higher ribosome count (Failmezger et al. 2018).

Although the system works well when expressing plasmids from E. coli, it could be shown that expression was much higher when plasmids prepared from V. natriegens were used. Possible explanations are differences in methylation patterns, resulting in degradation of the plasmid by the system (Failmezger et al. 2018). All this could be done by using protocols very similar to well established methods for other bacteria.

Cre- lox

The Cre- lox system was also successfully implemented and shown to work consistently with 600bp homology flanks (Weinstock et al. 2016) Recycling of selection markers, like antibiotic resistances, as well as the removal of incorporated genes becomes easy and reliable.

Also interesting for future protein related applications is, that a putative secretion signal was identified (Weinstock et al. 2016)

Fun facts:

The epithet natriegens was chosen after the realization that salt is needed for it to grow, natrie stemming from the Latin word natrium meaning sodium and the Latin verb egere, meaning to need.

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