MccV + Cvi (Microcin V + immunity protein)

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. Precursor MccV (prior to cleavage of the signal peptide) is shown in blue on the inner side of the membranes, and the final 'cleaved' MccV is shown outside, having been secreted from the cell. From Kim et al., 2023.

Microcin V (MccV) is the native cargo of the E. coli microcin V type I secretion system used in our Zone of Inhibition assays for microcin secretion. Cvi is the immunity protein for MccV which integrates into the cell membrane to help prevent toxicity of the microcin against the host bacterium. The antimicrobial activity of MccV is relatively well-documented (Kim et al., 2023), so we chose to use this microcin as a positive control when assessing the antimicrobial activity of our novel microcins. We included MccV and Cvi as a single part to ensure that host toxicity would not be an issue in our positive controls.

Composite Parts

Figure 2. The general schematic for our constitutive and inducible microcin + immunity protein assemblies with emphasis on the microcin + immunity protein part.

Constitutive MccV + Cvi expression plasmid (BBa_K4579046)

Design Notes

In order to make our microcin expression plasmid modular, we had to make the signal peptide modular (see Design Notes for our CvaC15 part for more info). This design decision meant that we also had to make several changes to the native E. coli cvaC and cvi genes—which encode microcin V (MccV) and its immunity protein Cvi respectively—when designing this part. In the E. coli genome, the coding sequence of cvi is upstream of the 5’ end of the coding sequence for cvaC. In order to utilize the modular CvaC15 signal peptide part and make it fuse to the N-terminus of the microcin, however, it was imperative that the microcin coding sequence be placed at the upstream end of the part so that the signal peptide could be transcribed and translated into a fusion protein with MccV or another microcin of choice. Because of this, we used two sets of primers to amplify cvaC and cvi independently with overhangs designed to ligate cvaC immunity protein to the end of the sequence. The immunity protein and MccV were assembled in the same assembly reaction in which they were cloned into the part plasmid vector using BsmBI to create the basic MccV + Cvi part.

Characterization

The MccV + Cvi part was characterized via a Zone of Inhibition assay (detailed on our Experiments page) to test the functionality of our modular microcin expression plasmid in conjunction with pSK01, as MccV has documented antimicrobial activity against E. coli and should show a zone if our system actually functions as expected (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 modular design 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 by our team from plasmid pSKP00 created by Kim et al.

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_K4579009