CvaAB - Type I secretion system proteins

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. This figure depicts the positions of CvaA and CvaB in the membrane and how they help facilitate secretion of peptides containing a signal peptide sequence (CvaC15 or BBa_K4579008) at their N-terminus. From Kim et al., 2023.

This is a Type 3 part that includes the coding sequences for the genes cvaA and cvaB. CvaA and CvaB are membrane proteins essential to the Gram-negative microcin V (MccV) type I secretion system, as their function is to help transport microcins outside the cell. A schematic of how this process works at the molecular level (created by Kim et al.) can be found in Figure 2 of our Project Description page.

Design Notes

Disclaimer: This is a Type IIS compatible version of the sequence we have used. We currently have on hand an equivalent non-compatible version of this sequence containing an illegal SapI site.

The original sequence of this part contained an illegal BsaI site as well as an illegal SapI site. Because our Golden Gate Assembly method utilizes BsaI to construct composite parts from different types of basic parts, we made a silent mutation to the coding sequence of CvaAB in order to remove the illegal BsaI restriction site. The site was near the start of the coding sequence of CvaB, so we designed 2 sets of primers to amplify sections of this part on either side of the illegal BsaI site. One of the primers was specifically designed with BsmBI overhangs that would generate a silent mutation in the restriction site GAGACC to convert it to GAAACC, retaining the amino acid residue (glutamate) encoded at that position. These PCR products were then assembled together scarlessly into a basic part entry vector using BsmBI.

The version of this part that we have on hand does not currently contain the silent mutation that removes the SapI site, as our assembly method only utilizes BsmBI and BsaI enzymes. However, we have not assembled or characterized this part because the pSK01 secretion system plasmid has proven sufficient for the needs of our assays and functioned in our chassis (Kim et al., 2023). As such, we could repeat the process described above for the removal of the BsaI site in order to mutate out the SapI site. In the sequence listed in the registry, a single base mutation has been made to convert the GCTCTTC SapI restriction site to GCTCTTT, changing the codon TTC to TTT and retaining the phenylalanine residue encoded at that position.

Characterization

All parts uploaded to the registry by our team, including this one, have been sequence confirmed.

Our team has not assembled this part into a composite part. Because we have been using the MccV signal peptide on our microcin expression plasmids, we performed all of our assays using the MccV secretion system genes cvaA and cvaB on the pSK01 plasmid which contains these genes under control of a constitutive promoter. This part was made in the event that the team wanted to place the secretion system genes under inducible control or otherwise change the surrounding genetic context of the CvaAB coding sequence.

Source

The sequences for cvaA and cvaB were amplified from secretion system plasmid pSK01 (Kim et al., 2023). These genes originate in E. coli.

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
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1715
Illegal EcoRI site found at 2382
Illegal PstI site found at 643
Illegal PstI site found at 856
Illegal PstI site found at 1733
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1715
Illegal EcoRI site found at 2382
Illegal PstI site found at 643
Illegal PstI site found at 856
Illegal PstI site found at 1733
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1715
Illegal EcoRI site found at 2382
Illegal BamHI site found at 270
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1715
Illegal EcoRI site found at 2382
Illegal PstI site found at 643
Illegal PstI site found at 856
Illegal PstI site found at 1733
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1715
Illegal EcoRI site found at 2382
Illegal PstI site found at 643
Illegal PstI site found at 856
Illegal PstI site found at 1733
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K4579007