Part:BBa_K4579002

P_LuxB promoter + RBS

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 part consists of the P_LuxB promoter upstream of a ribosome binding site and hammerhead ribozyme (HHRz) sequence. This promoter can be bound by LuxR (BBa_K4579028) which acts as an activator that binds to the promoter when bound by the inducer molecule OC6, allowing for the selective induction of transcription in cells containing both P_LuxB and the LuxR gene. This part can be used as a Type 2 part in the BTK/YTK standard.

Design Notes

When designing our inducible promoter parts, we chose to use the YFP expression individual sensor plasmids from the E. coli ‘Marionette’ paper as PCR templates (Meyer et al., 2019) as these contain inducible promoters and their regulatory transcription factors controlling expression of YFP on a standardized backbone. Due to the homology of the backbone between these plasmids, we created universal primers that allowed us to amplify the promoter from any one of the sensor plasmids. One big design complication was the fact that the YFP-expressing sensor plasmids all contained a BsaI site in the region just upstream of the promoter where one of the universal primers would bind. We designed our primers to create a single point mutation in order to mutate out this illegal BsaI site.

Characterization

This table summarizes which of the basic type 2 promoter parts we created and characterized by assembling into a level 1 composite part and running the following fluorescence experiments on.

Short Description	Basic Part ID	Composite Part (with GFP, Regulator and Terminator)
PTet* promoter + RBS	BBa_K4579000	BBa_K4579061
PTac promoter + RBS	BBa_K4579001	BBa_K4579058
PLuxB promoter + RBS	BBa_K4579002	None
PCymRC promoter + RBS	BBa_K4579003	None
PBAD promoter + RBS	BBa_K4579004	None
PVanCC promoter + RBS	BBa_K4579005	BBa_K4579059
PCin promoter + RBS	BBa_K4579006	BBa_K4579060

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Characterization Assays

Figure 3. This displays the dynamic range of all the inducible promoters we were able to characterize in E. Coli DH5α. + indicates the presence of inducer in the overnight culture and – indicates a lack of inducer in the overnight culture. A mean of all three fluorescence readings divided by mean OD600 were plotted on a bar graph. Standard deviation of readings was used to create error bars.

To test each of the inducible promoter systems we incorporated them with GFP into composite parts (BBa_K4579058, BBa_K4579059, BBa_K4579060, BBa_K4579061). The parts were sequence confirmed and subsequently transformed into DH5α. Inducers were added using the following volumes (LacI: 50 µL of 0.1 M IPTG, VanR: 5 µL of 100 mM vanillic acid, CinR: 5 uL of 10 mM OHC14, TetR: 18 µL of a ~54 µM stock). These cultures were then loaded into a clear backed 96 well plate (in triplicate) and into a plate reader. A single fluorescence reading was taken using Ex: 485, Em: 535 (Elston et al., 2023).

Figure 4. This displays the dynamic range of all the inducible promoters we were able to characterize in P. Agglomerans with the T1SS. + indicates the presence of inducer in the overnight culture and – indicates a lack of inducer in the overnight culture. A mean of all three fluorescence readings divided by mean OD600 were plotted on a bar graph. Standard deviation of readings was used to create error bars.

In order to test our inducible promoters in a chassis organism with a secretion system we transformed our inducible GFP expression plasmids into a P. Agglomerans strain with a T1SS. We opted to not use the LacI regulatory system (BBa_K4579058) since it had high levels of expression in the absence of inducer. 5 ml induced and uninduced overnight cultures were created per each strain. Inducers were added using the following volumes (VanR: 5 uL of 100 mM vanillic acid, CinR: 5 uL of 10 mM OHC14, TetR: 18 uL of a ~54 uM stock). These cultures were then loaded into a clear backed 96 well plate (in triplicate) and into a plate reader. A single fluorescence reading was taken using Ex: 485, Em: 535 (Elston et al., 2023).

Source

This part was sourced from pAJM.474 on AddGene (https://www.addgene.org/108526/) (Meyer et al., 2018). pAJM.474 contains YFP under inducible control by pLuxB and LuxR.

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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]