Part:BBa_K4579032

CinR activator

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 is a Type 4 part that includes a terminator upstream of a transcriptional unit that constitutively expresses a regulatory transcription factor. This part can be used in conjunction with the Type 2 P_Cin promoter part to regulate transcription of a Type 3 (or Type 3p+3q) coding sequence determined by the user. In the absence of OHC14, transcription of the coding sequence is in a net ‘off’ state. When OHC14 is added, it binds to CinR and binds it to P_Cin promoter. This turns transcription of the Type 3 coding sequence to the ‘on’ state. The terminator at the 5’ end of this part’s sequence marks the end of the preceding transcriptional unit started with the Type 2 promoter and Type 3 coding sequence parts. The remainder of this part’s sequence is the constitutively active transcriptional unit for CinR.

Composite Parts

Figure 2. The general schematic for our inducible microcin and GFP expression assemblies with emphasis on the transcriptional regulator. Although this example contains an immunity protein sequence, not all of our inducible microcin expression parts include an immunity protein.

• CinR regulated Mcc04 + IP expression plasmid (BBa_K4579052)
• CinR regulated Mcc04 expression plasmid (BBa_K4579053)
• CinR regulated GFP expression plasmid (BBa_K4579060)

Design Notes

When designing our transcriptional regulator 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 regulator from any one of the sensor plasmids.

Characterization

​​Below is the fluorescence characterization data for the four inducible promoter-regulator systems that we tested in composite parts using a GFP reporter gene (BBa_K4579035). The table below lists the inducible promoters within our parts collection, their basic part numbers, and their composite part numbers with the GFP reporter. Below that, the first figure shows fluorescence/OD600 data for the characterization of our inducible GFP assemblies in E. coli DH5α. This first experiment was done as a proof of concept to test whether our inducible GFP assemblies were functional. After confirming functionality of the assemblies by measuring levels of fluorescence under induced and non-induced conditions, we moved on to testing the assemblies in Pantoea agglomerans PNG92-11 in order to determine whether these inducible promoter systems are functional in Pantoea. This data confirms the functionality of our inducible promoter systems in Pantoea.

This table summarizes which of the basic type 4 regulatory gene with terminator 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)
TetR repressor	BBa_K4579026	BBa_K4579061
LacI repressor	BBa_K4579027	BBa_K4579058
LuxR activator	BBa_K4579028	None
CymR repressor	BBa_K4579029	None
AraC activator + AraE transporter	BBa_K4579030	None
VanR repressor	BBa_K4579031	BBa_K4579059
CinR activator	BBa_K4579032	BBa_K4579060

Characterization Assays

Figure 3. This displays the dynamic range of all the inducible promoters with their respective regulatory genes 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 with their respective regulatory genes 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 with their respective regulatory genes 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 transcriptional regulator part was amplified from pAJM.1642 using PCR before being integrated into a basic part plasmid for use in our assemblies. pAJM.1642 contains YFP under inducible control by P_Cin and CinR.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 875
  Illegal XhoI site found at 938
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 675
  
• 1000
  COMPATIBLE WITH RFC[1000]