Part:BBa_K4579025

Mcc04 + 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

This is a Type 3q part containing a putative, novel microcin identified using the program cinful (Cole et al., 2022) which uses bioinformatics and alignment analysis to identify potential microcins from the inputted genomes of bacteria. This part also contains the coding sequence for a potential immunity protein associated with this specific microcin. The coding sequence of the microcin is located upstream of the immunity protein coding sequence, with an intermediate ribosome binding site (RBS) separating the two.

The function of the microcin is to ideally inhibit growth of pathogenic species of Pantoea in our assays, and the function of the immunity protein is to help prevent the microcin from inhibiting the growth of the chassis expressing it.

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 Mcc04 + immunity protein expression plasmid (BBa_K4579043)
• CinR regulated Mcc04 + IP expression plasmid (BBa_K4579052)
• VanR regulated Mcc04 + IP expression plasmid (BBa_K4579054)
• TetR regulated Mcc04 + IP expression plasmid (BBa_K4579056)

Design Notes

We used cinful to predict and select potential microcins from genomes of bacteria related to our potential target pathogens. To adapt our putative novel microcins to our cloning scheme, we first removed the native signal peptide sequence from the beginning of each microcin so that it could become fused with our system’s signal peptide (BBa_K4579008) when assembled and expressed. Once the signal peptide sequence was removed, we obtained the original nucleotide sequence for each microcin by using the sequence start and stop positions provided in the cinful output to locate the sequence of the microcin within the genome. We then added flanking sequences on either side of each microcin that would add the necessary BsmBI and BsaI restriction sites for our cloning scheme (detailed in Figure 3 of our Parts page). The final sequences of microcins minus the signal peptide plus flanking restriction sites were then synthesized commercially as Gene Fragments by Twist Biosciences for use in our digestion-ligation reactions. For this part, we also added a putative immunity protein downstream of the microcin with an intervening ribosome binding site (RBS) to ensure that translation occurred properly.

Characterization

Characterization of our Mcc04 + immunity protein part was done by testing this part in a composite part (BBa_K4579043) in which the microcin and immunity protein are both constitutively expressed.

Figure 1. Zone of inhibition plate with Pantoea ananatis PNA 97-1R lawn as ‘prey’ against E. coli DH5α strain containing Mcc04 and associated immunity protein expressing plasmid as the ‘predator’. Controls include empty chassis and strain containing microcin expressing plasmid but not secretion system plasmid.

Figure 2. 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)

Source

This microcin was identified from the genome of Pantoea vagans PaVv9 using cinful. The immunity protein was identified from the genome of the same strain using the criteria described in the Results page of our website.

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., 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.
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 PstI site found at 128
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 128
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 128
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 128
  Illegal AgeI site found at 61
  
• 1000
  COMPATIBLE WITH RFC[1000]