Part:BBa_K4579060

CinR regulated GFP 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.

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

These are type 234 parts compatible with Golden Gate Assembly and contain a promoter, GFP (gfpmut3) followed by a regulatory gene and a terminator. The promoter and regulatory genes were derived from the E. coli ‘Marionette’ paper (Meyer et al., 2019) and GFP from the Bee Toolkit (Leonard et al., 2018). These parts were developed to characterize our inducible promoters via taking a fluorescence reading of both induced and uninduced overnight cultures. This composite part was nested within the pBTK1028 backbone sourced from the Bee Toolkit (Leonard et al., 2018). Plasmids were assembled using Golden Gate BsaI Assembly protocol.

This part contains the P_Cin promoter in front of GFP followed by the CinR activator gene and a terminator.

Design Notes

This part gained an illegal SpeI site due to the way that our basic part BioBricks were assembled. The specific overhangs used in this assembly caused the formation of an unexpected SpeI site at the junction of the promoter+RBS part and the GFP coding sequence part.

However, this is a terminal-stage part simply made to test our inducible promoter systems using GFP expression in various organisms, it is not meant for additional assembly. However, if one wanted to utilize this sequence for additional cloning, the single nucleotide T scar between the promoter+RBS and coding sequence can be altered to a C to create a legal junction site between these two BioBrick parts.

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.

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_VanCC and VanR.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 356
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 257
  Illegal SpeI site found at 356
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1957
  Illegal XhoI site found at 2020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 356
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 356
  Illegal NgoMIV site found at 1757
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 374