Evaluation Vector with PxylA to screen for protein specific secretion efficiency
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 247
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1354
Illegal AgeI site found at 1348
- 1000Illegal BsaI site found at 266
As stated on the main page of this part, 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 (Figure 1, A). However, on not X-Gal containing agar plates, the red color of the RFPsyn2 will be visible. This applies to colonies not carrying lacZα, too (Figure 1, B). E. coli colonies carrying neither lacZα nor RPFsyn2 will stay whitish as common E. coli colonies (Figure 1, C). By applying this setup, successfully transformed E. coli colonies can be identified easily.
As our project was based on the Gram-positive model organism Bacillus subtilis, we decided to use a previously well-evaluated B. subtilis vector as source for our Evaluation Vector: the integrative vector pBS1C1 (Radeck et al., 2013). 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. (For a detailed description of the original vector features please have a look at Radeck et al., 2013)
A confirmed BsaI restriction free version of the vector pBS1C was cut with EcoRI and XbaI to insert the xylose inducible promoter PxylA (Radeck et al., 2013) which was prior amplified using the following set of primers:
Following the amplification, we digested the PCR product with EcoRI and BsaI (resulting in an XbaI overhang) to maintain the BioBrick prefix in front of the promoter.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 PlacI 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 following set of primers:
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-PxylA backbone, which was prior opened using BsaI (resulting in an XbaI overhang) and PstI (Figure 2).
The final construct of our EV was then cut with EcoRI and PstI to be cloned into the pSB1C3 backbone and verified by sequencing prior to the submission to the partsregistry.
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. PubMed