CvaC15 - Signal peptide

Introduction

The 2023 UT Austin iGEM Team’s modular microcin expression parts collection includes parts necessary for engineering a bacterial chassis to secrete microcins, a type of small antimicrobial peptide. Our team has specifically designed parts to engineer a modular two-plasmid system that facilitates extracellular secretion of microcins by the chassis. One plasmid contains the microcin with a signal peptide sequence that indicates to the cell that the microcin is to be secreted. The other plasmid (pSK01) is from the literature (Kim et al., 2023) and contains genes for the proteins CvaA and CvaB, which are necessary to secrete small peptides using the E. coli microcin V (MccV) type I secretion system (T1SS) shown in Figure 2 of our Project Description.

Our parts collection includes a a selection of promoter (Type 2), coding sequence (Type 3), and terminator/regulatory gene (Type 4) parts that can be easily assembled to express microcins either constitutively or under inducible control. This allows for the modular engineering of microcin expression plasmids containing various microcins that can undergo extracellular secretion when used in conjunction with the secretion system plasmid pSK01.

Figure 1. Basic parts categorized by their BTK/YTK part type. Type 3p and 3q parts assemble as if they were a single Type 3 part.

Our basic and composite parts follow the Bee Toolkit/Yeast Toolkit standard of Golden Gate assembly (Lee et al., 2015; Leonard et al., 2018). Our assembly method involves the use of BsmBI digestion-ligation to create basic parts which can then be further digested with BsaI and ligated to form composite parts. The BTK/YTK standard includes part type-specific prefix and suffix overhangs generated by BsaI for each part, and these overhangs are NOT included in their sequences in the registry. For reference, our standard’s part type-specific overhangs are listed in Figure 2 on our Parts page.

Categorization

Basic parts

Promoters (Type 2) – Seven inducible promoters selected due to their relatively high dynamic range (Meyer et al., 2019) and apparent functionality in a variety of Proteobacteria (Schuster & Reisch, 2021), and one constitutive CP25 promoter (Leonard et al., 2018).
Coding Sequences (Type 3) – Signal peptide + microcin fusion coding sequences, a green fluorescent protein gene, and secretion system genes cvaA and cvaB which are together referred to as CvaAB.
Terminators/Regulatory Genes (Type 4) – An rpoC terminator plus a collection of seven regulatory genes, each associated with one of our seven inducible promoters.

Composite parts

Constitutive Microcin Expression Assemblies - Assemblies of microcins (some with immunity proteins) with a constitutive CP25 promoter and rpoC terminator. These function alongside pSK01 in a two-plasmid secretion system, and we use these two-plasmid systems to assess if our novel microcins are effective inhibitors of pathogenic targets.
Inducible GFP Expression Assemblies – Assemblies of GFP under the control of various inducible promoter systems. These were used to assess the dynamic range of our inducible promoter systems.
Inducible Microcin Expression Assemblies – Assemblies of select microcins under the control of an inducible promoter system.

Usage and Biology

Figure 2. Schematic of the microcin V type I secretion system in E. coli. The signal peptide (SP) is shown in orange at the N-terminus of Precursor MccV in the figure. From Kim et al., 2023.

This is a Type 3p part that contains the nucleotide sequence coding for the CvaC15 signal peptide. CvaC15 is a peptide derived from the N-terminus of E. coli microcin V (MccV). When fused to the N-terminus of a peptide such as a microcin, this signal peptide sequence enables the recognition of that peptide by MccV secretion system proteins shown in Figure 2. The Type 3 coding part for secretion system proteins CvaA and CvaB can be found at BBa_K4579007. Upon recognition by CvaB, the signal peptide is cleaved off, and the peptide cargo is exported from the cell (Kim et al., 2023). A schematic of how this process works at the molecular level can be seen in Figure 2 above, created by Kim et al. This part is included in composite parts BBa_K4579039 – BBa_K4579057.

Figure 3. The general schematic for our constitutive and inducible microcin assemblies with emphasis on the signal peptide. Although this example lacks an immunity protein, some of our microcin expression composite parts include an immunity protein.

Design Notes

All known microcins have a signal peptide amino acid sequence at their N-terminus, but this sequence differs depending on the specific variants of the secretion system genes within the microcin’s bacterium of origin. CvaC15 is the specific signal peptide recognized by the type I secretion system proteins encoded on the secretion plasmid pSK01 (Kim et al., 2023) that we use in conjunction with our microcin plasmids. We made CvaC15 as its own part in order to make the system modular, allowing any microcin to be engineered downstream of this signal peptide sequence on the microcin expression plasmid. By doing so, any microcin (or microcin + immunity protein) part can be swapped in or out of the system with the confidence that it will always be fused to the type I secretion system-compatible signal peptide CvaC15 when translated.

For the signal peptide to function properly in the secretion system, the microcin must be fused downstream of the signal peptide domain such that the two glycine residues at the C-terminus end of the signal peptide are in-frame with the amino acids of the microcin. These two glycines are the site of binding and cleavage by the cognate secretion system proteins that facilitate microcin export (Kim et al., 2023). We designed custom overhangs at the 3’ end of the signal peptide sequence and 5’ end of the microcin part sequences that would allow them to ligate scarlessly in a manner that places the nucleotides encoding the double glycine residue in frame with the microcin’s coding sequence.

We designed the BsaI restriction sites and part type-specific overhangs of CvaC15 such that the last nucleotide in the first glycine codon and the entire second glycine codon became the suffix for this part and the prefix of our microcin parts. When a microcin is assembled with the signal peptide, the microcin sequence “completes” the signal peptide sequence.

Characterization

The signal peptide part was characterized via a Zone of Inhibition assay (detailed on our Experiments page) to test microcin secretion using MccV as a positive control, as MccV has documented antimicrobial activity against E. coli (Kim et al., 2023). We specifically used the constitutive MccV + Cvi expression composite part BBa_K4579046 in conjunction with secretion plasmid pSK01 (Kim et al., 2023) in our assays to test the effectiveness of our signal peptide to enable secretion of a microcin with known activity against E. coli. For results from our MccV + Cvi expression composite part, see the Characterization section of BBa_K4579046.

Source

This part comes from E. coli and was cloned from microcin expression plasmid pSKP00 (Kim et al., 2023).

References

Cole, T. J., Parker, J. K., Feller, A. L., Wilke, C. O., & Davies, B. W. (2022). Evidence for widespread class II microcins in Enterobacterales Genomes. Applied and Environmental Microbiology, 88(23), e01486-22.
Kim, S. Y., Parker, J. K., Gonzalez-Magaldi, M., Telford, M. S., Leahy, D. J., & Davies, B. W. (2023). Export of Diverse and Bioactive Small Proteins through a Type I Secretion System. Applied and Environmental Microbiology, 89(5), e00335-23.
Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS Synthetic Biology, 4(9), 975-986.
Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., Kar, S., Davies, B. W., Ellington, D. E., Moran, N. A., & Barrick, J. E. (2018). Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids. ACS Synthetic Biology, 7(5), 1279-1290.
Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196-204.
Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Research, 49(12), 7189-7202.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K4579008