Part:BBa_K4579008
CvaC15 - Signal peptide
Introduction
The 2023 UT Austin iGEM Team’s Parts Collection includes a multitude of parts necessary for engineering bacteria to secrete microcins, a type of small antimicrobial peptide. Specifically, our team has designed parts that allow us to engineer a modular Biobrick-friendly version of an existing two-plasmid microcin secretion system1 that secretes putative novel microcins predicted by bioinformatics analysis.2 The first plasmid—the ‘microcin’ plasmid—contains the microcin and a signal peptide, while the second plasmid—the ‘secretion system’ plasmid—contains genes for two proteins of the E. coli microcin V type I secretion system (T1SS) machinery collectively referred to as CvaAB. Our parts can be easily assembled into transcriptional units to express any of our current 13 novel microcins (and potentially other small peptides1) either constitutively or under inducible control.
In our assembly schema, each basic part begins and ends with a distinct sequence of 4 nucleotides according to the part type derived from the syntax of the Bee Toolkit (BTK)3:
Promoters, whether inducible or constitutive, are designed to function as Type 2 parts.
The signal peptide is designed to function as a Type 3p part, while microcin or [microcin + immunity protein] parts are designed to function as Type 3q parts. Together, a Type 3p and Type 3q part form Type 3 parts in the BTK syntax.
Our team designed the overhangs to connect Type 3p to Type 3q parts, and these part types are not present in the original Bee Toolkit.
Terminators and [terminator + inducer-regulated transcription factor] parts are designed to function as Type 4 parts.
The various parts we have created can be assembled into microcin plasmid constructs with any other parts following the BTK syntax, allowing for the creation of flexible and modular designs by future iGEM teams that choose to work with microcins, secretion systems, or Golden Gate Assembly constructs. Additionally, we created a CvaAB part to allow for the recreation of the secretion system plasmid under a different promoter, origin, or selective marker depending on the needs at hand.
Characterization
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 syntax derived from the Yeast Toolkit (YTK) syntax.4 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
Two-Plasmid Secretion System Machinery – CvaC15 and CvaAB: These parts are necessary for the two-plasmid secretion system to function, regardless of what microcin or other peptide is being secreted using the system. In the BTK syntax, CvaAB is a Type 3 part, and CvaC15 is a Type 3p part.
Inducible Promoters – Marionette promoters: A collection of seven inducible promoters selected due to their functionality in a variety of Proteobacteria as shown by Schuster and Reisch in their 2021 paper “A plasmid toolbox for controlled gene expression across the Proteobacteria.” Each promoter also includes a hammerhead ribozyme (HHRz) to insulate the part from self-degradation.
Microcins or [microcin + immunity protein] units – MccV+Cvi and all novel microcins from cinful. Regulatory Genes – Marionette regulatory genes: These parts include a terminator upstream of the regulatory gene transcriptional unit to complete the microcin or microcin+immunity protein transcriptional unit.
Composite parts
Constitutive Microcin/[Microcin+Immunity Protein] Expression Assemblies
Inducible Promoter Characterization Assemblies – Assemblies of green fluorescent protein (GFPmut3) under the control of various inducible promoter + regulator pairs.
Inducible Microcin Expression Assemblies – Assemblies of microcins under the control of inducible promoter + regulator pairs. We have currently created such assemblies for two microcins:
Mcc04: A putative microcin identified from the genome of Pantoea vagans PaVv9 by cinful, a bioinformatics tool designed for microcin identification as described by Cole et al. in the 2022 paper “Evidence for Widespread Class II Microcins in Enterobacterales Genomes.” Microcin V: derived from E. coli and previously characterized in literature, was used as a positive control for secretion assays, as it is known to effectively target certain strains of E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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