At a Glance
Peptidosomes in combination with Bacillus subtilis offer a perfect platform for enhanced protein overproduction by the means of efficient protein secretion provided through B. subtilis and the easy purification due to the physical separation of bacteria and the end-product in the supernatant facilitated by the Peptidosomes. Naturally, B. subtilis is a strong secretion host and in order to take full advantage of this great potential it is necessary to evaluate all possible combinations of the B. subtilis’ secretion signal peptides and the proteins of interest. Therefore, we developed the Evaluation Vector (EV) which is a powerful genetic tool containing a multiple cloning site (MCS) specifically designed to easily exchange translational fusions composed of the desired protein and a secretion signal peptide.
Tool development and their proper evaluation are core aspects of Synthetic Biology. In our project EncaBcillus one main idea was to establish Peptidosomes with encapsulated bacteria as efficient protein overproduction platform. We took advantage of B. subtilis’ ability to efficiently secrete proteins into its environment in order to increase overall yields and to simplify the purification of the desired proteins.
Therefore, we developed a general expression Evaluation Vector (EV) with easily exchangeable units: I) allowing the replacement of the promoter (which drives the system) and II) a multiple cloning site enabling to work with translationally fused composite parts. In our case, a typical composite part consists of a signal peptide (for secretion in B. subtilis) and a protein of interest.
In summary, our EV was designed to fulfill the following distinct features:
- Exchangeable promoter region
- Insertion of basic or composite parts as expression units
- Fulfilling the RFC10 and RFC25 BioBrick standard
- Easy cloning and screening procedure in Escherichia coli
As our project is based on the Gram-positive model organism B. subtilis, we decided to use a previously well-evaluated B. subtilis vector as source for our Evaluation Vector: the integrative vector pBS1C1.  In brief, the vector has the following features for cloning in E.coli: an ori of replication and the bla gene mediating resistance against ampicillin. The B. subtilis specific part of the vector contains the multiple cloning site (MCS) in RFC10 standard, a cat cassette providing resistance against chloramphenicol and flanking regions needed for integration into the amyE locus. After integration into α-amylase, the resulting disruption of the native gene leads to a loss of this enzymatic activity, thereby making it a vector easy to screen for by performing a starch test for positive integration events. (For a detailed description of the original vector features please have a look at  and our Design section of the EV.)
As described above, we chose to modify the backbone of pBS1C1  to create our new Evaluation Vector (EV), by engineering the multiple cloning site (MCS) according to the scheme below (Figure 1).
Our team developed a unique multiple cloning site for the Evaluation Vector. The distinct features of the Evaluation Vector allow for easy insertion of both, a promoter and two basic or composite parts while providing an easy cloning and screening procedure. We evaluated and proved the applicability of the system multiple times and are sure, that this special multiple colng site can be of great value for future cloning.
|||Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7, 29.|