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| | + | <h1>Vibrio natriegens, a review.</h1> |
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| − | <i><u>
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| − | <h1>Vibrio natriegens</h1>
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| − | </i>
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| − |
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| − |
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| − | , a review.</h1>
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| − | <div class="collapsible">
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| − | <div class="btn_expand">Ecology</div>
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| − | <div class="content">
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| − | <i> V. natriegens </i> has some amazing abilities. By understanding them as adaptations, it becomes quite apparent that
| |
| − | <i>
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| − | V. natriegens
| |
| − | </i>
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| − | is itself first and foremost a product of its environment. Therefore, getting a better understanding of its ecology helps explain some of these capabilities.
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| − |
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| | | | |
| − | <p>
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| − | 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,
| |
| − | <i>
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| − | V. natriegens
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| − | </i>
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| − | 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
| |
| − | <a href="http://www.microbiologyresearch.org/docserver/fulltext/micro/116/2/mic-116-2-295.pdf?expires1538914706&id=id&accname=guest&checksum=F87669F5F1195DDDD75B002444425E85" ><abbr title="Adenine Nucleotide Pools During Starvation of Beneckea Natviegens."
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| − | >(Nazly 1980).</abbr>
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| − |
| |
| − | </a>
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| − | Furthermore, it has the ability to quickly draw available phosphorous from its environs and store them in polyphosphate bodies
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| − | <a href="http://www.nrcresearchpress.com/doi/10.1139/m87-101" ><abbr title="Growth, Elemental Composition, and Formation of Polyphosphate Bodies in Vibrio Natriegens Cultures Shifted from Phosphate-Limited to Phosphate-Pulsed Media."
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| − | >(Nissen <i> et al. </i> 1987)</abbr>
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| − |
| |
| − | </a>
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| − | for later utilization. Yet another way in which <i>
| |
| − | V. natriegens
| |
| − | </i> 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/17416432" ><abbr title="Production of Poly-β-Hydroxybutyrate (PHB) by Vibrio Spp. Isolated from Marine Environment."
| |
| − | >(Chien <i> et al. </i> 2007).</abbr>
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| − |
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| − | </a>
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| − |
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| − | </p>
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| − | <p>
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| − | From the well oxygenated upper layer of mud down to the anoxic layers, <i>
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| − | V. natriegens
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| − | </i> is able to grow in many surroundings. These extremes are challenges to which <i> V. natriegens </i> found solutions. At the surface of the mud, as well as in open waters, the bacteria are exposed to strong <dfn data-info="ultra violet"> UV </dfn> radiation. As a response to this, the <dfn data-info="desoxyribonucleic acid"> DNA </dfn> damage repair systems are highly active
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/21046343" ><abbr title="Identification of Vibrio Natriegens UvrA and UvrB Genes and Analysis of Gene Regulation Using Transcriptional Reporter Plasmids"
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| − | >(Simons <i> et al. </i> 2010),</abbr>
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| − |
| |
| − | </a>
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| − | with significantly elevated expression levels of some parts of the system compared to
| |
| − | <i>
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| − | E. coli
| |
| − | </i>
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| − | , thus increasing <dfn data-info="desoxyribonucleic acid"> DNA </dfn> sequence integrity. By switching to fermentation, growth can be maintained under anaerobic conditions, allowing the colonization of the lower strata in the mud.
| |
| − | </p>
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| − |
| |
| − |
| |
| − | <i>
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| − | V. natriegens
| |
| − | </i>
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| − | 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/11607653" ><abbr title="N2 Fixation in Marine Heterotrophic Bacteria: Dynamics of Environmental and Molecular Regulation."
| |
| − | >(Coyer <i> et al. </i> 1996)</abbr>
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| − |
| |
| − | </a>
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| − | under anaerobic conditions.
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| − |
| |
| − |
| |
| − | <p>
| |
| − | Unsurprisingly,
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
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| − | has its own predators in its native habitat: Bacteriophages
| |
| − | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC380186/pdf/applmicro00040-0188.pdf" ><abbr title="Isolation of Bacteriophages of the Marine Bacterium Beneckea Natriegens from Coastal Salt Marshes"
| |
| − | >(Zachary 1974).</abbr>
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| − |
| |
| − | </a>
| |
| − | Studies in the 70ies found a link between different phages and the prevailing environmental conditions
| |
| − | <a href="http://www.nrcresearchpress.com/doi/10.1139/m78-053" ><abbr title="An Ecological Study of Bacteriophages of Vibrio Natriegens"
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| − | >(Zachary 1978)</abbr>
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| − |
| |
| − | </a>
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| − | . 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/938035" ><abbr title="Physiology and Ecology of Bacteriophages of the Marine Bacterium Beneckea Natriegens: Salinity."
| |
| − | >(Zachary 1976).</abbr>
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| − |
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| − | </a>
| |
| − | Intriguingly, that opens the possibility of creating genetic parts derived from these phages, applicable to future work with
| |
| − | <i>
| |
| − | V. natriegens,
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| − | </i>
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| − | analogous to the T7 system in
| |
| − | <i>
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| − | E. coli.
| |
| − | </i>
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| − | On the other hand, one study could show, that the phage carrying the toxins associated with V. cholerae could not replicate in V. natriegens
| |
| − | <a href="https://www.biorxiv.org/content/early/2016/06/12/058487" ><abbr title="Vibrio Natriegens, a New Genomic Powerhouse."
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| − | >(Lee <i> et al. </i> 2016),</abbr>
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| − |
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| − | </a>
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| − | further showing its harmlessness towards humans.
| |
| − | </p>
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| − | <p>
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| − | In contrast to that, some strains of
| |
| − | <i>
| |
| − | Vibrio natriegens
| |
| − | </i>
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| − | seem to be predators themselves. Confirmed as being pathogenic in several marine crustacea, most notably in the swimming crab
| |
| − | <i>
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| − | Portunus trituberculatus,
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| − | </i>
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| − | in which
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| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
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| − | can lead to mortalities up to 85%
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| − |
| |
| − | <a href="http://doi.wiley.com/10.1111/jwas.12305" ><abbr title="Isolation and Molecular Identification of Vibrio Natriegens from Diseased Portunus Trituberculatus in China"
| |
| − | >(Bi <i> et al. </i> 2016).</abbr>
| |
| − | </a>
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| − | This is especially problematic since
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| − | <i>
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| − | P. trituberculatus
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| − | </i>
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| − | 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 <i>
| |
| − | V. natriegens
| |
| − | </i> are pathogenic to crustaceans, but it is well known that many Vibrio species cause opportunistic infections in crustacea.
| |
| − | </p>
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| − | <p>
| |
| − | On a more positive note, a recent study could show that the sponge
| |
| − | <i>
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| − | Aplysilla rosea
| |
| − | </i>
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| − | develops a more diverse microbiome when exposed to
| |
| − | <i>V. natriegens </i>
| |
| − | <a href="http://www.nature.com/articles/s41598-018-30295-y" ><abbr title="A Controlled Aquarium System and Approach to Study the Role of Sponge-Bacteria Interactions Using Aplysilla Rosea and Vibrio Natriegens."
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| − | >(Mehbub <i> et al. </i> 2018).</abbr>
| |
| − | </a>
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| − |
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| − |
| |
| − | It can be speculated that this helps to protect the sponge from pathogenic microbes that then find it harder to colonize its surface.
| |
| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − |
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| − | <div class="collapsible">
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| − | <div class="btn_expand">Metabolism </div>
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| − | <div class="content">
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| − |
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| − | </p>
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| − | <p>
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| − | A diverse, well-balanced metabolism allows a bacterium to grow in many conditions, while simultaneously making it interesting for biotechnological applications.
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| − | <i>
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| − | V. natriegens
| |
| − | </i>
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| − | can utilize a wide range of substrates
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/28887417" ><abbr title="High Substrate Uptake Rates Empower Vibrio Natriegens as Production Host for Industrial Biotechnology"
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| − | >(Hoffart <i> et al. </i> 2017),</abbr>
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| − |
| |
| − | </a>
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| − | as well as grow in the absence of oxygen while being faster than other bacteria under the same conditions.
| |
| − | </p>
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| − | <p>
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| − | 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
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| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/29042298" ><abbr title="Metabolism of the Fast-Growing Bacterium Vibrio Natriegens Elucidated by 13C Metabolic Flux Analysis"
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| − | >(Long <i> et al. </i> 2017).</abbr>
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| − |
| |
| − | </a>
| |
| − | By tracking 13C labeled carbon through the intricate network that constitutes
| |
| − | <i>
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| − | V. natriegens
| |
| − | </i>
| |
| − | metabolism, they were able to gain insight. Fore one, they found the it to be very similar to the well-studied
| |
| − | <i>
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| − | E. coli
| |
| − | </i>
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| − | metabolism. It has al the mayor 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
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
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| − | compared to only 21% in
| |
| − | <i>
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| − | E. coli,
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| − | </i>
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| − | the presence of an enzyme for the decarboxylation of oxalate
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| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/29042298" ><abbr title="Metabolism of the Fast-Growing Bacterium Vibrio Natriegens Elucidated by 13C Metabolic Flux Analysis.">
| |
| − | (Long <i> et al </i> 2017).
| |
| − | </a>
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| − |
| |
| − | </p>
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| − | <p>
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| − | 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/28887417" ><abbr title="High Substrate Uptake Rates Empower Vibrio Natriegens as Production Host for Industrial Biotechnology."
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| − | >(Hoffart <i> et al. </i> 2017).</abbr>
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| − |
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| − | </a>
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| − | 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
| |
| − | <i>
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| − | E. coli
| |
| − | </i>
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| − | and
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| − | <i>
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| − | C. glutamicum
| |
| − | </i>
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| − | production-strains by a factor of 9 to 13 times
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/28887417" ><abbr title="High Substrate Uptake Rates Empower Vibrio Natriegens as Production Host for Industrial Biotechnology."
| |
| − | >(Hoffart <i> et al. </i> 2017).</abbr>
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| − |
| |
| − | </a>
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| − | 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.
| |
| − | </p>
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| − | <p>
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| − | 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/13888946" ><abbr title="Pseudomonas Natriegens, a Marine Bacterium with a Generation Time of Less than 10 Minutes."
| |
| − | >(Eagon 1962).</abbr>
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| − |
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| − | </a>
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| − |
| |
| − | </p>
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| − | <p>
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| − | The dependence on Na+ ions for its metabolic activity conveys a form of natural biocontainment, inhibiting growth in accidentally released
| |
| − | <i>
| |
| − | V. natriegens</i>
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/4327612" ><abbr title="Influence of Na+ on Synthesis of Macromolecules by a Marine Bacterium.">(Webb and Payne 1971).</abbr>
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| − |
| |
| − | </a>
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| − |
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| − | The high salt content of the LBv2 media also presents possible contaminants with a barrier, reducing the likelihood of airborne microbes to take hold.
| |
| − | </p>
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| − | <p>
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| − | Recently, a study revealed that
| |
| − | <i>
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| − | V. natriegens,
| |
| − | </i>
| |
| − | surprisingly, is the most effective producer of selenium nanoparticles yet described
| |
| − | <a href="http://www.nature.com/articles/s41598-017-16252-1" ><abbr title="Speeding up Bioproduction of Selenium Nanoparticles by Using Vibrio Natriegens as Microbial Factory."
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| − | >(Fern`ndez-Llamosas <i> et al. </i> 2017).</abbr>
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| − |
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| − | </a>
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| − | These particles with a diameter from 100-400<dfn data-info="nanometer, 10^-9m">nm</dfn> 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,
| |
| − | <i>
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| − | V. natriegens
| |
| − | </i>
| |
| − | exceptionally high resistance to selenite suggests possible applications in bioremediation of contaminated water and soils.
| |
| − | </p>
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| − | <p>
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| − | <strong>
| |
| − | Working with
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | </strong>
| |
| − | </p>
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| − | <p>
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| − | <dfn data-info="American Type Culture Collection, identical to DSM 759">ATCC</dfn> 14048 is the most commonly used strain of
| |
| − | <i>
| |
| − | V. natriegens.
| |
| − | </i>
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| − | Most results from earlier studies were generated from this strain. We recommend using <dfn data-info="American Type Culture Collection, identical to DSM 759">ATCC</dfn> 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
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| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
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| − | >(Weinstock <i> et al. </i> 2016)</abbr>
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| − |
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| − | </a>
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| − | so you won´t miss out on its incredible doubling time.
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| − | </p>
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| − | <p>
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| − | Stemming from subtropical regions,
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | has not the same tolerance to low temperatures that we are used to from
| |
| − | <i>
| |
| − | E. coli,
| |
| − | </i>
| |
| − | but it is still much more resilient to cold than
| |
| − | <i>
| |
| − | B. subtilis.
| |
| − | </i>
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| − | At the root of that lies the low catalytic activity of
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | native catalase at low temperatures, which is then unable to detoxify reactive oxygen species. To address that problem, scientists from <dfn data-info="Synthetic Genomic Solutions">SGI</dfn> 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
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| − |
| |
| − | </a>
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| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | When working with <i>V. natriegens </i> <dfn data-info="American Type Culture Collection, identical to DSM 759">ATCC</dfn> 14048, no special precautions have to be taken since it has been shown that no known pathogenicity associated genes were present
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
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| − | Also, there is continued record of research since 1958 with not a single documented incident of a human infection with
| |
| − | <i>
| |
| − | V. natriegens.
| |
| − | </i>
| |
| − |
| |
| − | </div>
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| − | </div>
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| − |
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| − |
| |
| − | <div class="collapsible">
| |
| − | <div class="btn_expand"> Molecular Biology</div>
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| − | <div class="content">
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | Lately, a new branch of microbiologists developed an interest in
| |
| − | <i>
| |
| − | V. natriegens.
| |
| − | </i>
| |
| − | Their main focus lies in establishing state of the art methods and bringing
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | to its full potential. We see our project as part of this movement and wish to make
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | easily accessible to researchers. One groundbreaking paper
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
| |
| − | established several methods to make
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | genetically accessible, as well as characterizing some central genetic parts. This paved the way for more research into applying its potential.
| |
| − | </p>
| |
| − | <p>
| |
| − | If you want to introduce genetic material into
| |
| − | <i>
| |
| − | V. natriegens,
| |
| − | </i>
| |
| − | 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
| |
| − | <i>
| |
| − | E. coli
| |
| − | </i>
| |
| − | 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 (
| |
| − | <a href="https://www.biorxiv.org/content/early/2016/06/12/058487" ><abbr title="Vibrio Natriegens, a New Genomic Powerhouse."
| |
| − | >(Lee <i> et al. </i> 2016;</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | > Weinstock <i> et al. </i> 2016).</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | <p>
| |
| − | A fully sequenced genome is also a prerequisite for many studies. Currently, several sequences are available
| |
| − | <a href="https://www.biorxiv.org/content/early/2016/06/12/058487" ><abbr title="Vibrio Natriegens, a New Genomic Powerhouse"
| |
| − | > (Lee <i> et al. </i> 2016;</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/23969053" ><abbr title="Draft Genome Sequence of the Fast-Growing Bacterium Vibrio natriegens"
| |
| − | >Maida <i> et al. </i> 2013),</abbr>
| |
| − |
| |
| − | </a>
| |
| − | one of them generated by us. The genome has a size of approximately 5.17 <dfn data-info="mega bases, 10^9 bp">Mb</dfn>. Automated annotation was performed, revealing more of the genomic structure, as well as enabling the reconstruction of the codon usage profile
| |
| − | <a href="https://www.biorxiv.org/content/early/2016/06/12/058487" ><abbr title="Vibrio Natriegens, a New Genomic Powerhouse"
| |
| − | >(Lee <i> et al. </i> 2016).</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | <p>
| |
| − | The genome of
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | is, like in many species of the genus, split into two chromosomes. Chromosome one contains 3.24 <dfn data-info="mega bases, 10^9 bp">Mb</dfn>, chromosome two 1.92 <dfn data-info="mega bases, 10^9 bp">Mb</dfn>.
| |
| − | </p>
| |
| − | <p>
| |
| − | <strong>
| |
| − | Plasmids, Promoter, resistance
| |
| − | </strong>
| |
| − | </p>
| |
| − | <p>
| |
| − | Usually, a big hurdle when changing your chassis organism is a very basic question: Will my constructs still work? Do I need different <dfn data-info="origen of replication">ori</dfn>? 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
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
| |
| − | 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
| |
| − | <i>
| |
| − | E. coli.
| |
| − | </i>
| |
| − | Promoter commonly used in
| |
| − | <i>
| |
| − | E. coli
| |
| − | </i>
| |
| − | also lead to expression in
| |
| − | <i>
| |
| − | V. natriegens.
| |
| − | </i>
| |
| − | 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 <dfn data-info="RBS">ribosomal binding sites</dfn>.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | and shown to drive protein expression
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
| |
| − | One way of generating even stronger, phage derived promoters could be using
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | derived phage expression systems.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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
| |
| − | <i>
| |
| − | E. coli.
| |
| − | </i>
| |
| − | We measured the
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | specific concentrations and made them available on our strain engineering page.
| |
| − | </p>
| |
| − | <p>
| |
| − | 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.
| |
| − | </p>
| |
| − | <p>
| |
| − | <strong>
| |
| − | MuGENT - Recombineering
| |
| − | </strong>
| |
| − | </p>
| |
| − | <p>
| |
| − | Investigations into alternative genetic tools were also undertaken, most notably MuGENT and Recombineering.
| |
| − | </p>
| |
| − | <p>
| |
| − | MuGENT takes advantage of
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | natural competence by inducing the TfoX system. The TfoX system, which is known from many Vibrio species, enables uptake and genomic integration of <dfn data-info="deoxyribonucleic acid">DNA</dfn> parts via homologous recombination
| |
| − | <a href="http://doi.wiley.com/10.1111/j.1462-2920.2010.02250.x" ><abbr title="Natural Transformation of Vibrio Fischeri Requires TfoX and TfoY"
| |
| − | >(Pollack-Berti 2010).</abbr>
| |
| − |
| |
| − | </a>
| |
| − | In most cases, growth on chitin was identified as the trigger which activated the system. Regrettably, the trigger is not known for
| |
| − | <i>
| |
| − | V. natriegens.
| |
| − | </i>
| |
| − | Using this system, a group of researchers characterized this system in
| |
| − | <i>
| |
| − | V. natriegens,
| |
| − | </i>
| |
| − | 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
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/28571309" ><abbr title="Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio Natriegens."
| |
| − | > (Dalia <i> et al. </i> 2017).</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | <p>
| |
| − | Recombineering takes advantage of the fact that short <dfn data-info="deoxyribonucleic acid">DNA</dfn> 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 <dfn data-info="base pairs">bp</dfn>. Having this system available in
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | means that generating new strains and gene variants is easy and quickly implemented
| |
| − | <a href="https://www.biorxiv.org/content/early/2017/04/26/130088" ><abbr title="Vibrio Natriegens, a New Genomic Powerhouse"
| |
| − | >(Lee <i> et al. </i> 2017).</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | <p>
| |
| − | <strong>
| |
| − | Cell Free Protein-expression Systems (CFPS)
| |
| − | </strong>
| |
| − | </p>
| |
| − | <p>
| |
| − | Very recently, several groups have published their work about cell free protein expression systems (CFPS) in
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | <a href="https://www.frontiersin.org/article/10.3389/fmicb.2018.01146/full" ><abbr title="Cell-Free Protein Synthesis From Fast-Growing Vibrio Natriegens."
| |
| − | >(Failmezger <i> et al. </i> 2018;</abbr>
| |
| − |
| |
| − | </a>
| |
| − | </i>
| |
| − |
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/30107122" ><abbr title="Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio Natriegens."
| |
| − | >Des Soye <i> et al. </i> 2018;</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | <a href="http://pubs.acs.org/doi/10.1021/acssynbio.8b00222" ><abbr title="Establishing a Cell-Free Vibrio Natriegens Expression System."
| |
| − | > Wiegand <i> et al. </i> 2018).</abbr>
| |
| − |
| |
| − | </a>
| |
| − | Cell free systems offer the advantage of direct access to many cell components as well as fine control over the conditions in the reaction
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/22008973" ><abbr title="Cell-Free Protein Synthesis: Applications Come of Age."
| |
| − | > (Carlson <i> et al. </i> 2012).</abbr>
| |
| − |
| |
| − | </a>
| |
| − | While these are true for all <dfn data-info="Cell Free Protein-expression System">CFPSs</dfn>, the advantage of creating such a system from
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | is the much higher ribosome count
| |
| − | <a href="https://www.frontiersin.org/article/10.3389/fmicb.2018.01146/full" ><abbr title="Cell-Free Protein Synthesis From Fast-Growing Vibrio Natriegens."
| |
| − | >(Failmezger <i> et al. </i> 2018).</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | <p>
| |
| − | Although the system works well when expressing plasmids from
| |
| − | <i>
| |
| − | E. coli,
| |
| − | </i>
| |
| − | it could be shown that expression was much higher when plasmids prepared from
| |
| − | <i>
| |
| − | V. natriegens
| |
| − | </i>
| |
| − | were used. Possible explanations are differences in methylation patterns, resulting in degradation of the plasmid by the system
| |
| − | <a href="https://www.frontiersin.org/article/10.3389/fmicb.2018.01146/full" ><abbr title="Cell-Free Protein Synthesis From Fast-Growing Vibrio Natriegens."
| |
| − | >(Failmezger <i> et al. </i> 2018).</abbr>
| |
| − |
| |
| − | </a>
| |
| − | All this could be done by using protocols very similar to well established methods for other bacteria.
| |
| − | </p>
| |
| − | <p>
| |
| − | <strong>
| |
| − | Cre-
| |
| − | <i>
| |
| − | lox
| |
| − | </i>
| |
| − | </strong>
| |
| − | </p>
| |
| − | <p>
| |
| − | The Cre-
| |
| − | <i>
| |
| − | lox
| |
| − | </i>
| |
| − | system was also successfully implemented and shown to work consistently with 600bp homology flanks
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
| |
| − | Recycling of selection markers, like antibiotic resistances, as well as the removal of incorporated genes becomes easy and reliable.
| |
| − | </p>
| |
| − | <p>
| |
| − | Also interesting for future protein related applications is, that a putative secretion signal was identified
| |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/27571549" ><abbr title="Vibrio Natriegens as a Fast-Growing Host for Molecular Biology"
| |
| − | >(Weinstock <i> et al. </i> 2016)</abbr>
| |
| − |
| |
| − | </a>
| |
| − |
| |
| − | </p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </p>
| |
| − | <p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <p>
| |
| − | Fun facts:
| |
| − | </p>
| |
| − | <p>
| |
| − | The epithet <i> natriegens </i> was chosen after the realization that salt is needed for it to grow, <i>natrie</i> stemming from the Latin word natrium meaning sodium and the Latin verb <i>egere</i>, meaning to need.
| |
| − | </p>
| |
| − | <p><b>
| |
| − | </b>
| |
| − | <p><b>
| |
| − | <b>
| |
| − | <b>
| |
| − | <b>
| |
| − | <div class="collapsible">
| |
| − | <div class="btn_expand">Sources</div>
| |
| − | <div class="content">
| |
| − | <P>
| |
| − | </P>
| |
| − | Aiyar, Sarah E, Tamas Gaal, and Richard L Gourse. 2002. RRNA Promoter Activity in the Fast-Growing Bacterium Vibrio Natriegens.
| |
| − | <i>
| |
| − | Journal of bacteriology
| |
| − | </i>
| |
| − | 184(5): 1349-58. http://www.ncbi.nlm.nih.gov/pubmed/11844764 (August 23, 2018).
| |
| − |
| |
| − | <p>
| |
| − | Austin, Payne B, A Zachary, and R R Colwell. 2018. 28 International Association of Microbiological Societies
| |
| − | <i>
| |
| − | Recognition of Beneckea Natriegens (Payne et al.; Baumann et al.) as a Member of the Genus Vibrio, as Previously Proposed by Webb and Payne
| |
| − | </i>
| |
| − | . www.microbFiologyresearch.org (August 23, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Bi, Keran <i> et al. </i> 2016. Isolation and Molecular Identification of
| |
| − | <i>
| |
| − | Vibrio Natriegens
| |
| − | </i>
| |
| − | from Diseased
| |
| − | <i>
| |
| − | Portunus Trituberculatus
| |
| − | </i>
| |
| − | in China.
| |
| − | <i>
| |
| − | Journal of the World Aquaculture Society
| |
| − | </i>
| |
| − | 47(6): 854-61. http://doi.wiley.com/10.1111/jwas.12305 (September 29, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Carlson, Erik D, Rui Gan, C Eric Hodgman, and Michael C Jewett<dfn data-info="A fish is just a fish, and weather it is eaten depends much on the dish!">.</dfn> 2012. Cell-Free Protein Synthesis: Applications Come of Age.
| |
| − | <i>
| |
| − | Biotechnology advances
| |
| − | </i>
| |
| − | 30(5): 1185–94. http://www.ncbi.nlm.nih.gov/pubmed/22008973 (October 1, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | CHIEN, C <i> et al. </i> 2007. “Production of Poly-β-Hydroxybutyrate (PHB) by Vibrio Spp. Isolated from Marine Environment.”
| |
| − | <i>
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| − | Journal of Biotechnology
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| − | </i>
| |
| − | 132(3): 259–63. http://www.ncbi.nlm.nih.gov/pubmed/17416432 (September 22, 2018).
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| − | </p>
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| − | <p>
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| − | Coyer, J A, A Cabello-Pasini, H Swift, and R S Alberte. 1996. “N2 Fixation in Marine Heterotrophic Bacteria: Dynamics of Environmental and Molecular Regulation.”
| |
| − | <i>
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| − | Proceedings of the National Academy of Sciences of the United States of America
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| − | </i>
| |
| − | 93(8): 3575–80. http://www.ncbi.nlm.nih.gov/pubmed/11607653 (August 23, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Dalia, Triana N. <i> et al. </i> 2017. “Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in
| |
| − | <i>
| |
| − | Vibrio Natriegens
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| − | </i>
| |
| − | .”
| |
| − | <i>
| |
| − | ACS Synthetic Biology
| |
| − | </i>
| |
| − | 6(9): 1650–55. http://www.ncbi.nlm.nih.gov/pubmed/28571309 (September 30, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Delpech, Roger. 2001. “Using Vibrio Natriegetis for Studying Bacterial Population Growth, Artificial Selection, and the Effects of UV Radiation and Photo-Reactivation.”
| |
| − | <i>
| |
| − | Journal of Biological Education
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| − | </i>
| |
| − | 35(2): 93–97. http://www.tandfonline.com/doi/abs/10.1080/00219266.2000.9655749 (August 23, 2018).
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| − | </p>
| |
| − | <p>
| |
| − | EAGON, R G. 1962. “Pseudomonas Natriegens, a Marine Bacterium with a Generation Time of Less than 10 Minutes.”
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| − | <i>
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| − | Journal of bacteriology
| |
| − | </i>
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| − | 83(4): 736–37. http://www.ncbi.nlm.nih.gov/pubmed/13888946 (August 23, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Failmezger, Jurek, Steffen Scholz, Bastian Blombach, and Martin Siemann-Herzberg. 2018. “Cell-Free Protein Synthesis From Fast-Growing Vibrio Natriegens.”
| |
| − | <i>
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| − | Frontiers in Microbiology
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| − | </i>
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| − | 9: 1146. https://www.frontiersin.org/article/10.3389/fmicb.2018.01146/full (September 30, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Fernández-Llamosas, Helga <i> et al. </i> 2017. “Speeding up Bioproduction of Selenium Nanoparticles by Using Vibrio Natriegens as Microbial Factory.”
| |
| − | <i>
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| − | Scientific Reports
| |
| − | </i>
| |
| − | 7(1): 16046. http://www.nature.com/articles/s41598-017-16252-1 (September 28, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Hoffart, Eugenia <i> et al. </i> 2017. “High Substrate Uptake Rates Empower Vibrio Natriegens as Production Host for Industrial Biotechnology.”
| |
| − | <i>
| |
| − | Applied and environmental microbiology
| |
| − | </i>
| |
| − | : AEM.01614-17. http://www.ncbi.nlm.nih.gov/pubmed/28887417 (October 6, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Lee, Henry H <i> et al. </i> 2016. “Vibrio Natriegens, a New Genomic Powerhouse.”
| |
| − | <i>
| |
| − | bioRxiv
| |
| − | </i>
| |
| − | : 058487. https://www.biorxiv.org/content/early/2016/06/12/058487 (September 30, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Lee, Henry H, Nili Ostrov, Michaela A Gold, and George M Church. 2017. “Recombineering in Vibrio Natriegens.”
| |
| − | <i>
| |
| − | bioRxiv
| |
| − | </i>
| |
| − | : 130088. https://www.biorxiv.org/content/early/2017/04/26/130088 (September 30, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Long, Christopher P., Jacqueline E. Gonzalez, Robert M. Cipolla, and Maciek R. Antoniewicz. 2017. “Metabolism of the Fast-Growing Bacterium Vibrio Natriegens Elucidated by 13C Metabolic Flux Analysis.”
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| − | <i>
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| − | Metabolic Engineering
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| − | </i>
| |
| − | 44: 191–97. http://www.ncbi.nlm.nih.gov/pubmed/29042298 (October 5, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Maida, I. <i> et al. </i> 2013. “Draft Genome Sequence of the Fast-Growing Bacterium Vibrio Natriegens Strain DSMZ 759.”
| |
| − | <i>
| |
| − | Genome Announcements
| |
| − | </i>
| |
| − | 1(4). http://www.ncbi.nlm.nih.gov/pubmed/23969053 (August 23, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Mehbub, Mohammad F. <i> et al. </i> 2018. “A Controlled Aquarium System and Approach to Study the Role of Sponge-Bacteria Interactions Using Aplysilla Rosea and Vibrio Natriegens.”
| |
| − | <i>
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| − | Scientific Reports
| |
| − | </i>
| |
| − | 8(1): 11801. http://www.nature.com/articles/s41598-018-30295-y (September 29, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Miura, A <i> et al. </i> 1981. “Growth-Rate-Dependent Regulation of Ribosome Synthesis in E. Coli: Expression of the LacZ and GalK Genes Fused to Ribosomal Promoters.”
| |
| − | <i>
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| − | Cell
| |
| − | </i>
| |
| − | 25(3): 773–82. http://www.ncbi.nlm.nih.gov/pubmed/6793240 (September 22, 2018).
| |
| − | </p>
| |
| − | <p>
| |
| − | Mullenger, L., and Nijole R. Gill. 1973. “Vibrio Natriegens: A Rapidly Growing Micro-Organism Ideally Suited for Class Experiments.”
| |
| − | <i>
| |
| − | Journal of Biological Education
| |
| − | </i>
| |
| − | 7(5): 33–39. http://www.tandfonline.com/doi/abs/10.1080/00219266.1973.9653881 (August 21, 2018).
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