Difference between revisions of "Part:BBa K4579008"
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<partinfo>BBa_K4579008 short</partinfo>
 
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<h1>Introduction</h1>
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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 <i>E. coli</i> microcin V (MccV) type I secretion system (T1SS) shown in Figure 2 of our <html><a href="https://2023.igem.wiki/austin-utexas/description">Project Description.</a></html>
  
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===Introduction===
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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.
  
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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 Biobrick-friendly version of an existing two-plasmid microcin secretion system1 that secretes putative novel microcins predicted by bioinformatics analysis.2 The first plasmid—the ‘microcin’ plasmid—contains the microcin 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 13 novel microcins (and potentially other small peptides1) either constitutively or under inducible control.
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<html><center><img src=https://static.igem.wiki/teams/4579/wiki/parts-collection-by-type.jpeg style="width:900px;height:auto;"></center></html>
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<center><b>Figure 1.</b> <i>Basic parts categorized by their BTK/YTK part type. Type 3p and 3q parts assemble as if they were a single Type 3 part.</i> </center>
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In our assembly schema, each basic part begins and ends with a distinct sequence of 4 nucleotides according to the part type derived from the syntax of the Bee Toolkit (BTK)3:
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'''Promoters''', whether inducible or constitutive, are designed to function as Type 2 parts.  
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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 <html><a href=" https://2023.igem.wiki/austin-utexas/parts">Parts page</a></html>.  
  
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'''The signal peptide''' is designed to function as a Type 3p part, while '''microcin''' or [microcin + immunity protein] parts are designed to function as Type 3q parts. Together, a Type 3p and Type 3q part form Type 3 parts in the BTK syntax.
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<h1>Categorization</h1>
  
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Our team designed the overhangs to connect Type 3p to Type 3q parts, and these part types are not present in the original Bee Toolkit.  
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===Basic parts===
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<ul>
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<li><b>Promoters (Type 2)</b> – 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).</li>
  
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'''Terminators''' and [terminator + inducer-regulated transcription factor] parts are designed to function as Type 4 parts.  
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<li><b>Coding Sequences (Type 3)</b> – Signal peptide + microcin fusion coding sequences, a green fluorescent protein gene, and secretion system genes <i>cvaA</i> and <i>cvaB</i> which are together referred to as CvaAB.</li>
  
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<html><img src=https://static.igem.wiki/teams/4579/wiki/parts-collection-images/parts-collection-images/ggaoverhangs.png alt="Caption text." style="width:600px;height:auto;"></html>
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<li><b>Terminators/Regulatory Genes (Type 4)</b> – An <i>rpoC</i> terminator plus a collection of seven regulatory genes, each associated with one of our seven inducible promoters.</li>
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</ul>
  
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The various parts we have created can be assembled into microcin plasmid constructs with any other parts following the BTK syntax, allowing for the creation of flexible and modular designs by future iGEM teams that choose to work with microcins, secretion systems, or Golden Gate Assembly constructs. Additionally, we created a CvaAB part to allow for the recreation of the secretion system plasmid under a different promoter, origin, or selective marker depending on the needs at hand.
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===Composite parts===
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<ul>
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<li><b>Constitutive Microcin Expression Assemblies</b> - Assemblies of microcins (some with immunity proteins) with a constitutive CP25 promoter and <i>rpoC</i> 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.</li>
  
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===Characterization===
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<li><b>Inducible GFP Expression Assemblies</b> – Assemblies of GFP under the control of various inducible promoter systems. These were used to assess the dynamic range of our inducible promoter systems.</li>
  
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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 syntax derived from the Yeast Toolkit (YTK) syntax.4 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'''.
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<li><b>Inducible Microcin Expression Assemblies</b> – Assemblies of select microcins under the control of an inducible promoter system.</li>
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====Basic parts====
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</ul>
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'''Two-Plasmid Secretion System Machinery''' – CvaC15 and CvaAB: These parts are necessary for the two-plasmid secretion system to function, regardless of what microcin or other peptide is being secreted using the system.  
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In the BTK syntax, CvaAB is a Type 3 part, and CvaC15 is a Type 3p part.
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'''Inducible Promoters – Marionette promoters''': A collection of seven inducible promoters selected due to their functionality in a variety of Proteobacteria as shown by Schuster and Reisch in their 2021 paper “A plasmid toolbox for controlled gene expression across the Proteobacteria.” Each promoter also includes a hammerhead ribozyme (HHRz) to insulate the part from self-degradation.
 
  
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'''Microcins or [microcin + immunity protein] units''' – MccV+Cvi and all novel microcins from cinful.
 
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Regulatory Genes – Marionette regulatory genes: These parts include a terminator upstream of the regulatory gene transcriptional unit to complete the microcin or microcin+immunity protein transcriptional unit.
 
  
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====Composite parts====
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<h1>Usage and Biology</h1>
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'''Constitutive Microcin/[Microcin+Immunity Protein] Expression Assemblies'''
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'''Inducible Promoter Characterization Assemblies''' – Assemblies of green fluorescent protein (GFPmut3) under the control of various inducible promoter + regulator pairs.
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'''Inducible Microcin Expression Assemblies''' – Assemblies of microcins under the control of inducible promoter + regulator pairs. We have currently created such assemblies for two microcins:
 
  
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'''Mcc04''': A putative microcin identified from the genome of ''Pantoea vagans'' PaVv9 by cinful, a bioinformatics tool designed for microcin identification as described by Cole et al. in the 2022 paper “Evidence for Widespread Class II Microcins in Enterobacterales Genomes.”
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==Composite Parts==
  
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'''Microcin V''': derived from ''E. coli'' and previously characterized in literature, was used as a positive control for secretion assays, as it is known to effectively target certain strains of ''E. coli''. 
 
  
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<ul>
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<li>list item 1</li>
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<li>list item 2</li>
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<li>list item 3</li>
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</ul>
  
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===Usage and Biology===
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<h1>Design Notes</h1>
  
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CvaC15 is a signal peptide derived from the N-Terminus of the ''E. Coli'' native Microcin V. This part allows for proteins to be recognized by the gram-negative T1SS (CvaA/B) system we have utilized and is subsequently cleaved off during cargo export. 
 
  
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<h1>Characterization</h1>
  
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===References===
 
  
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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.
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<h1>Source</h1>
  
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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.  
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<h1>References</h1>
 +
<ol>
 +
<li>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. <i>Applied and Environmental Microbiology, 88</i>(23), e01486-22.</li>
  
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Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., ... & 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.  
+
<li>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. <i>Applied and Environmental Microbiology, 89</i>(5), e00335-23.</li>
  
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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.  
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<li>Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. <i>ACS Synthetic Biology, 4</i>(9), 975-986.</li>
  
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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.  
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<li>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. <i>ACS Synthetic Biology, 7</i>(5), 1279-1290.</li>
  
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Lou, C., Stanton, B., Chen, Y. J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137-1142.  
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<li>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. <i>Nature Chemical Biology, 15</i>(2), 196-204.</li>
 +
 
 +
<li>Schuster, L. A., & Reisch, C. R. (2021). A plasmid toolbox for controlled gene expression across the Proteobacteria. <i>Nucleic Acids Research, 49</i>(12), 7189-7202.</li>
 +
</ol>
  
 
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<span class='h3bb'>Sequence and Features</span>
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<h1>Sequence and Features</h1>  
 
<partinfo>BBa_K4579008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4579008 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
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<partinfo>BBa_K4579008 parameters</partinfo>
 
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Revision as of 04:21, 12 October 2023


CvaC15 - Signal peptide

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


Composite Parts

  • list item 1
  • list item 2
  • list item 3

Design Notes


Characterization


Source

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]