Short Description

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.

Background

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 [1]. 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 Radeck et al., 2013 and our Design section of the EV.)

Design

As described above, we chose to modify the backbone of pBS1C1 [1] to create our new Evaluation Vector (EV), by engineering the multiple cloning site (MCS) according to the scheme below (Figure 1).

At first, we removed a BsaI restriction enzyme site on the backbone of the vector by PCR based mutagenesis using primers TM3161 and TM3164 because it was interfering with our design. The confirmed BsaI free vector was then cut with EcoRI and XbaI to insert the Xylose inducible promoter P_xylA [1] wich was prior amplified using the primers iG17P051 and iG17P052 followed by digestion with EcoRI and BsaI (resulting in an XbaI overhang) to maintain the BioBrick prefix in front of the promoter. Next, we had to create an entirely new multiple cloning site (MCS): We synthesized a new RFP based on the sequence of the RFP found in the pSB1C3 backbone. The expression of this RFPsyn2 is still driven by the IPTG inducible P_lacI promoter but lacks any restriction enzyme sites interferring with the RFC25 standard.

Additionally, we added an AgeI restriction enzyme site downstream of the RFP coding sequence which is necessary for translational fusions. Furthermore, we amplified a lacZα fragment with AgeI and NgoMIV restriction enzyme sites upstream of the coding sequence and the RFC10 BioBrick standard as suffix using the primers iG17P055 and iG17P056.
Finally, we combined our new MCS, by ligating the digested RFPsyn2 (cut with XbaI and AgeI) with the lacZα fragment (cut with AgeI and PstI). This MCS was inserted into the pBS1C-P_xylA backbone, which was prior opened using BsaI (resulting in an XbaI overhang) and PstI (Figure 2). The final construct of our EV was verified by sequencing.

We decided to additionally also provide this MCS as a BioBrick. Therefore we cloned it into the pSB1C3 backbone (via EcoRI and PstI digest) and verified the construct by sequencing. It has been submitted to the parts registry under BBa_K2273107.

Detailed methods can be found in our protocol collection section. All primers used can be found in our primer collection table down below.

Primer collection table

Results

We constructed the Evaluation Vector (EV) to quickly screen for the secretion of a protein of interest. This vector contains a specifically designed MCS equipped with reporters to quickly identify positive replacements by insert integration (Figure 3). Additionally, this vector can be applied for the expression of any other fusion protein of interest regulated by a promoter of your choice. For more details on the application related with our project check out our Signal Peptide Toolbox and Secretion project.

Due to the unique setup of the MCS which provides easy access to powerful cloning, we provide the MCS as a part stored in the pSB1C3 backbone for the iGEM community (BioBrick BBa_K2273107).

As stated above in the Background section of the EV, we aimed for an easy cloning and screening procedure in our cloning host Escherichia coli. To accomplish that, we chose to set the construct RFPsyn2 as placeholder for the N-terminally fused protein and the gene lacZα for the C-terminally fused protein, respectively. Therefore, the blue color of lacZα carrying colonies and thereby X-Gal degrading colonies masks the red color of the RFPsyn2 on X-Gal containing agar plates. However, on not X-Gal containing agar plates, the red color of the RFPsyn2 will be visible. E. coli colonies carrying neither lacZα nor RPFsyn2 will stay whitish as common E. coli colonies (Figure 4). By applying this setup, successfully transformed E. coli colonies can be identified easily, as stated below in the standard operating procedure (SOP) protocol.

Based on our design, we established the following SOP protocol for cloning with the EV.

	SOP
1	Exchange of the promoter: Digest both, the provided EV and the new promoter using the restriction enzymes EcoRI and BsaI. Successfully transformed E. coli colonies on X-Gal containing agar plates stay blue. But on not X-Gal containing agar plates, successfully transformed E. coli colonies stay red.
2	Insertion of the C-terminally fused protein: Digest both, the EV from step 1 and the new gene of interest using the restriction enzymes NgoMIV and PstI. Successfully transformed E. coli colonies on X-Gal containing agar plates become red. But on not X-Gal containing agar plates, successfully transformed E. coli colonies stay red.
3	Insertion of the N-terminally fused protein: Digest the EV from step 2 using the restriction enzymes BsaI and NgoMIV BUT digest the gene of interest using the restriction enzymes XbaI and AgeI. Successfully transformed E. coli colonies on X-Gal containing agar plates become white. But on not X-Gal containing agar plates, successfully transformed E. coli colonies become white.
	We recommend to purify all digested products via gel extraction.

Additionally, a second SOP was tailored to explain the random integration of secretion signal peptides as the EV was evaluated in the course of our Signal Peptide Toolbox.

References

[1]

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.