Constitutive Mcc04 expression plasmid

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

This composite part is a transcriptional unit that constitutively expresses Mcc04, a novel microcin identified by the bioinformatics tool cinful, which is suspected to have antimicrobial activity against P. agglomerans PNG 92-11.

Characterization

This part was characterized using Zone of Inhibition assays (detailed on our Experiments page) that tested whether the incorporation of this microcin into our two-plasmid secretion system in our chassis would inhibit growth of a plant pathogenic strain of interest. Growth curve assays were also done to observe self-inhibition of growth of a pathogen that expresses the microcin. We determined from characterization of our constitutive expression assembly for MccV (BBa_K4579046), a microcin with known antimicrobial activity (Kim et al., 2023), that our engineered microcin expression plasmid does indeed produce an inhibitory effect when used with secretion plasmid pSK01 (Kim et al., 2023). This enabled us to move on to testing our modular microcin expression system with novel microcins like this one.

Figure 3. Backlit zone of inhibition plate with Pantoea ananatis PNA 97-1R lawn as ‘prey’ against E. coli DH5α strain containing this plasmid and pSK01 as the ‘predator’. Controls include empty chassis and strain containing microcin expressing plasmid but not secretion system plasmid. Small zone of inhibition (ZOI) was observed around the Mcc04 expressing and secreting strain. 

Figure 4. Backlit zone of inhibition plate with Pantoea allii PNA 200-100 lawn as ‘prey’ against E. coli DH5α strain containing this plasmid and pSK01 as the ‘predator’. Controls include empty chassis and strain containing microcin expressing plasmid but not secretion system plasmid. No zone of inhibition was observed around the Mcc04 expressing and secreting strain. 

Figure 5. Growth curves for pathogenic Pantoea strains transformed with this plasmid to observe self-inhibition of growth. The pathogenic Pantoea strains transformed with this plasmid were A) P. agglomerans PNG 92-11 B) P. allii PNA 200-100 C) P. ananatis LMG 2665. Mcc04 was observed to cause visible inhibition of P. agglomerans PNG 92-11. 

Figure 6. Two repeats of the growth curve assay done on two different days to show activity of P. agglomerans PNG 92-11 containing the modular microcin and immunity protein expressing assembly. Each faded curve is a replicate of an individual colony of the strain; there are 8 total replicates for each strain. The orange growth curve is the strain containing the microcin and immunity protein assembly and secretion system; blue growth curve is the strain containing only the microcin assembly and secretion system; grey growth curve is the strain containing microcin back bone and secretion system (negative control). 
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.</li>
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.

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Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 258
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 258
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 258
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 258
Illegal AgeI site found at 191
1000
COMPATIBLE WITH RFC[1000]

Part:BBa_K4579042