Part:BBa_K4579007

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 Link Text 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 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 in our collection categorized by BTK/YTK part type.

Our basic and composite parts follow the Bee Toolkit/Yeast Toolkit standard of Golden Gate assembly (Lee et al., 2015; Leonard et al., 2018). This standard includes type-specific prefix and suffix overhangs for each part, but these overhangs are NOT included in their sequences in the registry unless they include a section of the part—as seen in the Type 3 part prefix which includes the ATG start codon of the coding sequence. For reference, part type-specific prefix and suffix overhangs are listed in Figure 2 on our Parts Page.

Categorization

The basic parts that we developed to engineer a microcin-expressing two-plasmid system each fall into one of four categories listed below under the heading Basic Parts. Each part follows the Bee Toolkit (BTK) Golden Gate Assembly standard (Leonard et al., 2018) derived from the Yeast Toolkit (YTK) standard (Lee et al., 2015). Type-specific overhangs from this syntax can be added to the ends of any sequence intended to take on the function of that part type. Three categories of assemblies of our team’s basic parts alongside select parts from the Bee Toolkit are listed below under the heading Composite Parts.

Basic parts

1. Two-Plasmid Secretion System Machinery – CvaC15 signal peptide and CvaAB membrane proteins: These parts are necessary for the two-plasmid secretion system to function, regardless of what peptide is being secreted.
• In the language of our team’s adaptation of the BTK/YTK standard, cvaAB is a Type 3 part and cvaC15 is a Type 3p part.
2. Inducible Promoters – A collection of 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). Each of these parts also includes a ribosome binding site (RBS) and a hammerhead ribozyme (HHRz) in the 5' untranslated region to insulate gene expression levels from coding sequence effects on mRNA structure.
• In the language of our team’s adaptation of the BTK/YTK standard, these are Type 2 parts.
3. Microcin or Microcin+Immunity Protein coding sequences – All novel microcins that our team identified (some with immunity proteins) as well as the known microcin MccV + its associated immunity protein Cvi.
• In the language of our team’s adaptation of the BTK/YTK standard, these are Type 3q parts.
4. Regulatory Genes – A collection of seven regulatory transcription factor genes, each associated with one of the seven inducible promoters chosen for the reasons described above. These parts include a terminator upstream of the transcriptional unit such that this part completes the preceding microcin or microcin + immunity protein transcriptional unit.
• In the language of our team’s adaptation of the BTK/YTK standard, these are Type 4 parts.
5. BTK parts – Parts not previously found in the registry that originate from the Bee Toolkit created by Leonard et al. in 2018. These parts were not created by the UT Austin iGEM Team.
• These include pBTK107, a Type 2 CP25 constitutive promoter part, pBTK205, a Type 3 GFP coding sequence part, and pBTK300, a Type 4 rpoC terminator part.

Composite parts

1. Constitutive Microcin or Microcin+Immunity Protein Expression Assemblies - Assemblies of microcins under control of a constitutive CP25 promoter. These were the first composite parts created by our team, and we created them to assess whether our novel microcins would demonstrate effective inhibition of pathogenic targets when expressed constitutively.
2. Inducible Promoter Characterization Assemblies – Assemblies of green fluorescent protein (gfpmut3) under the control of various inducible promoter systems. These were used to analyze the ability of our inducible promoters and their regulators to produce an expression response in the presence of their respective inducer molecules.
3. Inducible Microcin Expression Assemblies – Assemblies of select microcins under the control of an inducible promoter system.

This part's categorization

cvaAB is a Type 3 part in the BTK/YTK standard and falls into the category of Two-Plasmid Secretion System Machinery basic parts.

Design Notes

[design]

Source

[source]

References

1. 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.
2. 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.
3. 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.
4. Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., ... & 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.
5. 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.
6. 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]