Revision as of 12:06, 12 October 2023

Constitutive Mcc04 + immunity protein 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 composite part is a transcriptional unit that constitutively expresses Mcc04—a novel microcin identified by the bioinformatics tool cinful—as well as its putative immunity protein. These two coding sequences are separated by a ribosome binding site (RBS) to ensure that translation of the parts occurs as intended. Mcc04 is suspected to have antimicrobial activity against Pantoea species, and the immunity protein is suspected to help prevent self-inhibition by the microcin against the host chassis.

Characterization

This part was characterized using both Zone of Inhibition assays (detailed on our Experiments page) and growth curves (see Experiments page). The Zone of Inhibition assays 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, and the growth curve assays were designed to determine if the microcin inhibited growth of the chassis itself. 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 and their putative immunity proteins.