Protein Secretion in B. subtilis
For the development of our project, we had to research the protein secretion pathways in Bacillus subtilis. As a contribution for future iGEM teams, we present our review, so that others can benefit from it. The Bacillus subtilis bacteria have the ability to overproduce a wide range of industrial enzymes. To accomplish this, a thorough study of the unique characteristics of B.subtilis' native protein secretory pathway to the extracellular medium is required [1].
The general secretion (Sec) pathway is B. subtilis' principal protein transportation route across the cytoplasmic membrane. This pathway sends proteins to the cytoplasmic membrane for insertion into or translocation over it [2]. In general, the Sec pathway is made up of Sec-dependent signal peptides that are followed by the moiety of mature proteins. In terms of overall structure, Sec signal peptides have a tripartite structure that includes a positively charged amino-terminal region, a hydrophobic core, and a polar carboxyl-terminal region that contains the signal peptidase recognition site [3]. Sec-dependent secretion has two distinct pathways: co-translational and post-translocation [2].
Another pathway is the Tat, which varies from the Sec in that it may transport completely folded proteins with a conserved region containing the twin-arginine motif in the signal peptide sequences. This pathway allows proteins to be secreted that would otherwise be too rapidly and tightly folded in the cytoplasm to be compatible with the first secretion system presented [4].
B. subtilis Protein Secretion Cassettes
The constructs would be built with the intent of studying different signal peptides for the secretion of proteins in a B. subtilis chassis and identifying the best one. To achieve this, the pBS1C plasmid backbone would be used and an insert sequence (Figure 1) designed [5]. The insert sequence contains two regulator sequences from the lac operon, the CAP binding protein site and the lac operator sequence. The CAP binding sequence binds the catabolite activator protein in the presence of cAMP and activates transcription for the lac promoter by binding to its specific DNA sites thus enhancing the ability of RNA polymerase to bind and initiate transcription [6]. On the other hand, the lac operon is responsible for blocking the transcription when the lac repressor is bound, however, in the presence of lactose, this repressor unbinds the lac operator and, thus, enables RNA polymerase to bind to the promoter. In most experiments, an analogue of lactose is used, IPTG (Isopropyl β-D-1-thiogalactopyranoside). The ribosome binding site in bacterial mRNA, and ensures the translation of proteins in B. subtilis. It is a rich A/G region, upstream of the start codon, ATG, of an mRNA. Translation initiation in bacteria requires RBS (Ribosome Binding Site), as it is responsible for the recruitment of the small bacterial ribosome (30S) and the translation initiation [6-7]. To allow for easy exchange of the signal peptide, restriction sites are placed for BamHI and Apal restriction enzymes upstream and downstream of the signal peptide sequence, respectively.
GFP is a protein in the jellyfish Aequorea victoria that exhibits green fluorescence when exposed to UV light. The protein has 238 amino acids, three of them (Numbers 65 to 67), form a structure that emits visible green, fluorescent light. In the jellyfish, GFP interacts with another protein, called aequorin, which emits blue light when added with calcium [8-9]. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm [9]. As such, GPF would have the function of a reporter protein in assays with the intent to measure the successful secretion of proteins like PETase and MHETase in a B. subtilis chassis. This protein’s sequence also is flanked by a standard Biobrick prefix and a suffix which would allow for the easy exchange from the reporter protein, GFP, to the insert of interest to the study. The construct would be then finalized by a rho-independent T7 terminator, T7Te, this region encodes for an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues which allows for the efficient transcription termination.
Figure 1: Schematic representation of the parts design for our Protein Secretion Cassettes.
This construct is designed with the intent to allow easy exchange of signal peptides and/or the gene of interest, and we have designed a total of 5 different ready to use cassettes, corresponding to 5 different signal peptides (links down below). Additionally, one can also change the signal peptide to experiment with another alternative sequence. To achieve this, one must digest the plasmid (with the construct), and the signal peptide sequence, with the restriction enzymes BamHI and ApaI. This would remove the existing signal peptide. In order to connect a new signal peptide sequence to the construct, the digested plasmid and new signal peptide sequence should be incubated with DNA ligase. To replace GFP by the gene of interest, in order to express the target protein, after selecting the best signal peptide, a similar procedure should be performed, however, the biobrick exchange system should be used instead.
Click on the links in the following list to access the maps of each individual protein secretion cassette:
References
- Anné, J., Economou, A. & Bernaerts, K. Protein Secretion in Gram-Positive Bacteria: From Multiple Pathways to Biotechnology. Curr. Top. Microbiol. Immunol. 404, 267–308 (2016).
- Chatzi, K. E., Sardis, M. F., Karamanou, S. & Economou, A. Breaking on through to the other side: protein export through the bacterial Sec system. Biochem. J. 449, 25–37 (2013).
- Rusch, S. L. & Kendall, D. A. Interactions That Drive Sec-Dependent Bacterial Protein Transport. Biochemistry 46, 9665–9673 (2007).
- Lee, P. A., Tullman-Ercek, D. & Georgiou, G. The Bacterial Twin-Arginine Translocation Pathway. Annu Rev Microbiol 60, 373–395 (2006).
- Radeck, Jara, et al. "The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis." Journal of biological engineering 7.1 (2013): 1-17.
- Hu, Yangbo, et al. "Ribosomal binding site switching: an effective strategy for high-throughput cloning constructions." PloS one 7.11 (2012): e50142.
- Omotajo, Damilola, et al. "Distribution and diversity of ribosome binding sites in prokaryotic genomes." BMC genomics 16.1 (2015): 1-8.
- Tsien, Roger Y. "The green fluorescent protein." Annual review of biochemistry 67.1 (1998): 509-544.
- Phillips, Gregory J. "Green fluorescent protein–a bright idea for the study of bacterial protein localization." FEMS microbiology letters 204.1 (2001): 9-18.
