Difference between revisions of "Team:Marburg/Parts"

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 the 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 our 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 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 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 have 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 TfoX expression 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 the V. cholerae Tfox is leading to competence in V. natriegens cells as well (Hayes et al. 2017).

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.

Since we are convinced, that E. coli will be replaced by V. natriegens in the next 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 TfoX expression (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.

Click here to download the protocol we used for natural transformation

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 (For more information see Strain Engineering results).