As best basic part we woud like to present <i>tfoX</i>, the inducer of natural competence in Vibrio species.
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Since scientific progress keeps accelerating in the past years, the fast production of results gains more and more importance. We are convinced that in the near future the community of synthetic biology will replace <i>E. coli</i> by the much faster growing organism <i>V.
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natriegens</i>. We strongly believe that our
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best basic part <i>tfoX</i> will support you in speeding up your research. Making this
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part accessible for everyone we would like to accelerate engineering biology and
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help you to make the world a better place.
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Tfox is known as the inducer of natural competence in some Vibrio species like <i>V. natriegens</i>. This ability of cells to take up free DNA is called competence. A special kind is the natural competence. In 1928, natural competence was first observed by Frederick Griffith even if he did not understand the genetics behind
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<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2167760/" target="blank"><abbr title="Griffith F, The significance of pneumococcal types. Journal of Hygiene. 27, Nr. 2. (1928) S. 113–59.">(Griffith<i>et al.</i>1928)</abbr></a>
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. Thanks to many more great scientists who did research in this area, we now know a lot about natural competence and the ability of some
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bacteria to take up free DNA.<br>
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In their natural environment, the regulator protein Tfox activates Type VI secretion systems (T6SSs) and allows bacteria to penetrate prey cells, leading to cell lysis and the release of DNA. The naturally competent cells can take up the free DNA from their environment and utilize it for genomic modifications by homologous recombination into their genome. Although much progresses has been made in this area, the molecular details of natural competence are not understood completely
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<a href="https://www.ncbi.nlm.nih.gov/pubmed/26442598" target="blank"><abbr title="Wu R., Zhao M., Li J., Gao H., Kan B., Liang W. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios. Sci Rep. (2015) 7;5:14921.">(Wu<i>et al.</i>2015)</abbr></a>
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For <i>V. cholerae</i>, it is known, that the expression of <i>tfoX</i> is induced by surface contact to chitin (Figure 1). Even though the <i>V. natriegens</i> genome is encoding a <i>tfoX</i> gene, its natural inducer is not known until now. However it is known, that the induction of expression of the <i>V. cholerae</i> <i>tfoX</i> is leading to competence in <i>V. natriegens</i> cells as well.
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<a href="https://www.ncbi.nlm.nih.gov/pubmed/28571309" target="blank"><abbr title="Dalia, T. N., C. A. Hayes, S. Stolyar, C. J. Marx, J. B. McKinlay and A. B. Dalia (2017). Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio natriegens. ACS Synth Biol 6(9): 1650-1655." ACS SynthBiol6(9):1650-1655.">(Hayes <i>et al.</i> 2017)</abbr></a>.
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<figure style="width: 95%;"><img style="display:block; margin:0 auto 0 auto; width:100%;" src="https://static.igem.org/mediawiki/2018/1/18/T--Marburg--tfoxonchitin.png" alt="Schematic figure on mode of action of TfoX.">
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<figcaption><b>Figure 1:</b> Schematic figure on mode of action of TfoX in <i>V. cholerae</i>. When cells get in contact with chitin, <i>tfoX</i> is expressed. Since TfoX is a positive regulator of natural competence, DNA from the environment is taken up into the cell.</figcaption>
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</figure>
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We compared the domains of TfoX with the domains of homologous proteins. These domains are listed below and in Figure 2.<p>
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<ul>
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<li><b>TfoX, N-terminal (TfoX_N):</b> N-terminal domain of TfoX found in Haemophilus influenza, also present in Sxy found in E. coli (inducer of natural competence)</li>
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<li><b>TfoX, C-terminal (TfoX_C):</b> C-terminal domain of TfoX found in Haemophilus influenza. This family corresponds to the C-terminal presumed domain of TfoX. The domain is found in association with the N-terminal domain in some, but not all members of this group, suggesting this is an autonomous and functionally unrelated domain.</li>
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<li><b>Coiled Coil region (CC):</b> Coiled Coil domain. This family is found in many proteins. Sometimes they are responsible for self-interaction, sometimes they are used to keep distance to other proteins.</li>
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</ul>
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<center><figure style="width: 70%;"><img style="display:block; margin:0 auto 0 auto; width:100%;" src="https://static.igem.org/mediawiki/2018/6/6f/T--Marburg--tfoxdomains.png" alt="Schematic figure on mode of action of TfoX.">
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<figcaption><b>Figure 1:</b> Schematic figure on mode of action of TfoX in <i>V. cholerae</i>. When cells get in contact with chitin, <i>tfoX</i> is expressed. Since TfoX is a positive regulator of natural competence, DNA from the environment is taken up into the cell.</figcaption>
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</figure></center>
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Since we are convinced, that <i>E. coli</i> will be replaced by <i>V. natriegens</i> in the next few years, we believe that the usage of natural transformation will become more significant. There are several advantages of this method in comparison to electroporation or heat shock mediated transformation. First, it is less stressful to the cells and second, you do not need to aliquot a thousands of chemically competent <i>E. colis</i> in Eppendorf cups every few weeks. If you are planning to do a transformation, the only thing to do is to inoculate the desired <i>V. natriegens</i> strain in a liquid culture, inducing the expression of <i>tfoX</i> (Figure 3). Additionally, modified <i>V. natriegens</i> strains can be produced much faster, due to the possibility to transform linear fragments for genomic modifications instead of wasting time with cloning plasmids. Even more linear fragments can be used for co-transformation, creating several genomic modifications at the same time
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<a href="https://www.ncbi.nlm.nih.gov/pubmed/28571309" target="blank"><abbr title="Dalia, T. N., C. A. Hayes, S. Stolyar, C. J. Marx, J. B. McKinlay and A. B. Dalia (2017). Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio natriegens. ACS Synth Biol 6(9): 1650-1655." ACS SynthBiol6(9): 1650-1655.">(Hayes <i>et al.</i> 2017)</abbr></a>
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.<br>
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In the future natural transformation could be utilized in combination with genome engineering methods like MAGE and CAGE, which rely on electroporation for <i>E.coli</i>, that could allow more rapid genome designs, such as complete codon reassignments.</p>
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<center><figure style="width: 60%;"><img style="display:block; margin:0 auto 0 auto; width:100%;" src="https://static.igem.org/mediawiki/2018/6/60/T--Marburg--naturaltransformationprotocol.svg" alt="Schematic figure on mode of action of TfoX." alt="Workflow of a natural transformation">
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<figcaption><b>Figure 3:</b>Workflow of a natural transformation.</figcaption>
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</figure></center>
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<p><a href="2018.igem.org/wiki/images/d/d5/T--Marburg--natural_transformation_protocol.pdf" target="blank">Click here to download the protocol we used for natural transformation</a></p>
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<p>Providing this basic part, we want to encourage the community of iGEM as well as all other scientists in the field of synthetic biology to start working with <i>V. natriegens</i>. Including <i>tfoX</i> into the modular cloning system of iGEM and the Marburg Collection we hope to usher in a new era of synthetic biology. We would love to see future iGEM teams using this basic part for further constructing their individual transcription unit variants in order to screen for the maximum efficiency for customized applications.</p>
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Using <i>tfox</i> in full transcriptional units, we were able to perform natural transformation with plasmids (Figure 4) as well as with linear DNA to create our strains with genomic modifications.
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<center><figure style="width: 50%;"><img style="display:block; margin:0 auto 0 auto; width:100%;" src="https://static.igem.org/mediawiki/2018/e/ed/T--Marburg--pYTKtransformation.svg" alt="Plates showing successful transformation of pYTK.">
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<figcaption><b>Figure 4:</b> Plates showing the successful transformation of plasmids using natural transformation. Left plate is showingtransformation of cells with pYTK. Right plate is showing negative control.</figcaption>
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</figure></center>
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Click here to learn more about how we used our best basic part in our project on the following sites in the Strain Engineering sub-project:<br>
As best basic part we woud like to present tfoX, the inducer of natural competence in Vibrio species.
Since scientific progress keeps accelerating in the past years, the fast production of results gains more and more importance. We are convinced that in the near future the community of synthetic biology will replace E. coli by the much faster growing organism V.
natriegens. We strongly believe that our
best basic part tfoX will support you in speeding up your research. Making this
part accessible for everyone we would like to accelerate engineering biology and
help you to make the world a better place.
Tfox is known as the inducer of natural competence in some Vibrio species like V. natriegens. This ability of cells to take up free DNA is called competence. A special kind is the natural competence. In 1928, natural competence was first observed by Frederick Griffith even if he did not understand the genetics behind
(Griffithet al.1928)
. Thanks to many more great scientists who did research in this area, we now know a lot about natural competence and the ability of some
bacteria to take up free DNA.
In their natural environment, the regulator protein Tfox activates Type VI secretion systems (T6SSs) and allows bacteria to penetrate prey cells, leading to cell lysis and the release of DNA. The naturally competent cells can take up the free DNA from their environment and utilize it for genomic modifications by homologous recombination into their genome. Although much progresses has been made in this area, the molecular details of natural competence are not understood completely
(Wuet al.2015)
.
For V. cholerae, it is known, that the expression of tfoX is induced by surface contact to chitin (Figure 1). Even though the V. natriegens genome is encoding a tfoX gene, its natural inducer is not known until now. However it is known, that the induction of expression of the V. choleraetfoX is leading to competence in V. natriegens cells as well.
(Hayes et al. 2017).
Figure 1: Schematic figure on mode of action of TfoX in V. cholerae. When cells get in contact with chitin, tfoX is expressed. Since TfoX is a positive regulator of natural competence, DNA from the environment is taken up into the cell.
We compared the domains of TfoX with the domains of homologous proteins. These domains are listed below and in Figure 2.
TfoX, N-terminal (TfoX_N): N-terminal domain of TfoX found in Haemophilus influenza, also present in Sxy found in E. coli (inducer of natural competence)
TfoX, C-terminal (TfoX_C): C-terminal domain of TfoX found in Haemophilus influenza. This family corresponds to the C-terminal presumed domain of TfoX. The domain is found in association with the N-terminal domain in some, but not all members of this group, suggesting this is an autonomous and functionally unrelated domain.
Coiled Coil region (CC): Coiled Coil domain. This family is found in many proteins. Sometimes they are responsible for self-interaction, sometimes they are used to keep distance to other proteins.
Figure 1: Schematic figure on mode of action of TfoX in V. cholerae. When cells get in contact with chitin, tfoX is expressed. Since TfoX is a positive regulator of natural competence, DNA from the environment is taken up into the cell.
Since we are convinced, that E. coli will be replaced by V. natriegens in the next few years, we believe that the usage of natural transformation will become more significant. There are several advantages of this method in comparison to electroporation or heat shock mediated transformation. First, it is less stressful to the cells and second, you do not need to aliquot a thousands of chemically competent E. colis in Eppendorf cups every few weeks. If you are planning to do a transformation, the only thing to do is to inoculate the desired V. natriegens strain in a liquid culture, inducing the expression of tfoX (Figure 3). Additionally, modified V. natriegens strains can be produced much faster, due to the possibility to transform linear fragments for genomic modifications instead of wasting time with cloning plasmids. Even more linear fragments can be used for co-transformation, creating several genomic modifications at the same time
(Hayes et al. 2017)
.
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.
Providing this basic part, we want to encourage the community of iGEM as well as all other scientists in the field of synthetic biology to start working with V. natriegens. Including tfoX into the modular cloning system of iGEM and the Marburg Collection we hope to usher in a new era of synthetic biology. We would love to see future iGEM teams using this basic part for further constructing their individual transcription unit variants in order to screen for the maximum efficiency for customized applications.
Using tfox in full transcriptional units, we were able to perform natural transformation with plasmids (Figure 4) as well as with linear DNA to create our strains with genomic modifications.
Figure 4: Plates showing the successful transformation of plasmids using natural transformation. Left plate is showingtransformation of cells with pYTK. Right plate is showing negative control.
Click here to learn more about how we used our best basic part in our project on the following sites in the Strain Engineering sub-project:
