Part:BBa_K4579052
CinR regulated Mcc04 + IP 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.
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 is an inducible type 234 transcriptional unit part that controls the inducible expression of the microcin Mcc04 and its immunity protein. This part contains the PCin promoter in front of Mcc04 and its immunity protein followed by a terminator and the transcriptional unit for the CinR activator gene. CinR activates transcription from the PCin promoter when bound by its inducer molecule OHC14. The goal of using inducible promoter systems with our microcins is to allow transcription of the microcin to be turned on or off at will, ideally preventing the chassis from suffering any adverse effects of constitutive microcin expression.
Characterization
We have not characterized our inducible promoter system + microcin assemblies, but all parts uploaded to the registry by our team, including this one, have been sequence confirmed.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 356
Illegal PstI site found at 534 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 257
Illegal SpeI site found at 356
Illegal PstI site found at 534 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1804
Illegal XhoI site found at 1867 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 356
Illegal PstI site found at 534 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 356
Illegal PstI site found at 534
Illegal NgoMIV site found at 1604
Illegal AgeI site found at 467 - 1000COMPATIBLE WITH RFC[1000]
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