Revision as of 04:08, 9 October 2023

CvaAB - Type I secretion system proteins

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 two-plasmid microcin secretion system that secretes putative novel microcins predicted by bioinformatics analysis. The first plasmid—the ‘microcin’ plasmid—contains the microcin (in some cases with its immunity protein) 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 novel microcins either constitutively or under inducible control.

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. Microcins 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

Constitutive Microcin/[Microcin+Immunity Protein] Expression Assemblies - Assemblies of microcins under control of a constitutive CP25 promoter.

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 select microcins under the control of an inducible promoter system.

Usage and Biology

Characterization

References

Sequence and Features