Parts
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), originating from Kim et al., 2023, 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 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. Created with BioRender.com.
Our basic and composite parts follow the Bee Toolkit/Yeast Toolkit (BTK/YTK) 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 below.
Figure 2. A schematic of our assemblies accompanied by a table of part type-specific overhangs, derived from the BTK/YTK standard. The sequence in the upper right depicts restriction sites and overhangs utilized in our Golden Gate Assembly process. Type-specific overhangs in the upper right sequence are labeled in red (prefix) and purple (suffix). Created with BioRender.com.
As mentioned previously, a two-stage Golden Gate Assembly process was utilized to generate our composite parts, the first stage of which involved the creation of basic parts. PCR products and gBlocks contained flanking BsaI sites that would produce part type-specific overhangs at their ends when digested, and these sites were in turn flanked by BsmBI sites for ligation into the backbone part pBTK1001 from the Bee Toolkit (Leonard et al., 2018). These basic parts could then be digested with BsaI and ligated together in a reaction with pBTK1028 (BBa_K4579033) from the Bee Toolkit, an mScarlet dropout backbone that is classified as a Type 56781 part in the BTK/YTK standard (Leonard et al., 2018). This was how we created our composite parts, and the overall process is depicted in Figure 3.
Figure 3. The process by which we assembled our basic and composite parts using Golden Gate Assembly. Basic parts were assembled using BsmBI digestion into a GFP dropout backbone known as pBTK1001; composite parts were assembled using BsaI digestion on basic parts into pBTK1028, an mScarlet dropout backbone part (BBa_K4579033). Created with BioRender.com.
The basic parts that we developed each fall into one of the categories listed under Basic Parts. Each of these follows the BTK/YTK standard mentioned in the Introduction. Type-specific overhangs from this standard 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 are listed under Composite Parts.
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 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.
Figure 4. Our modular microcin expression plasmid (a composite part) categorized by basic part constituents. Parts of the same type may be interchanged due to the use of standardized type-specific overhangs. Created with BioRender.com.
We nominate a portion of our parts collection for the Best Parts Collection Award because these parts assemble and function together to create the crux of our project: a modular microcin expression plasmid. These parts are also modular due to their part type-specific overhangs, meaning that parts of the same type can be swapped, making the process of microcin secretion customizable. The modular nature of our selected parts means that they could be helpful for future iGEM teams that want to use customizable secretion systems in their research, both in pathogenic and non-pathogenic strains. The collection includes:
- Our two most promising novel, putative microcins. One of these microcins is included in two of our basic parts, one with its associated immunity protein and one without: BBa_K4579025, BBa_K4579014, BBa_K4579012
- The signal peptide associated with the secretion system plasmid pSK01 (Kim et al., 2023) which can be fused to the N-terminus of microcins to make them compatible with the secretion system machinery: BBa_K4579008
- Our characterized inducible promoters and associated transcriptional regulators that can help regulate microcin production: BBa_K4579000, BBa_K4579001, BBa_K4579005, BBa_K4579006, BBa_K4579026, BBa_K4579027, BBa_K4579031, BBa_K4579032
We have tested all of these parts in some capacity (see Characterization sections for each part) and received positive results, which boosts our confidence in these parts. These parts are representative of our modular microcin expression system as well as the impact we hope to make on the agricultural community by providing a sustainable solution to tackling bacterial plant pathogens. In terms of the iGEM community, we hope that these parts provide a framework for future iGEM teams to incorporate modular secretion systems into their research.
This contains all of our parts except the backbone of our assemblies (BBA_k4579033):
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Constitutive Mcc04 + immunity protein expression plasmid
Constitutive microcin + immunity protein expression
Type 234
Composite
Constitutive MccV + Cvi expression plasmid
Constitutive microcin + immunity protein expression
Type 234
Composite
Constitutive MccE03 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE07 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE08 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE10 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccX07 expression plasmid
Constitutive microcin expression
Type 234
Composite
CinR Regulated Mcc04 + IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite
VanR regulated Mcc04+IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite
TetR regulated Mcc04 + IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite
Resgistry Name
Short Description
Category
Part Type
Basic/Composite
Constitutive Mcc04 + immunity protein expression plasmid
Constitutive microcin + immunity protein expression
Type 234
Composite
Constitutive MccV + Cvi expression plasmid
Constitutive microcin + immunity protein expression
Type 234
Composite
Constitutive MccE03 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE07 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE08 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccE10 expression plasmid
Constitutive microcin expression
Type 234
Composite
Constitutive MccX07 expression plasmid
Constitutive microcin expression
Type 234
Composite
CinR Regulated Mcc04 + IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite
VanR regulated Mcc04+IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite
TetR regulated Mcc04 + IP expression plasmid
Inducible microcin + immunity protein expression
Type 234
Composite