Difference between revisions of "Part:BBa K4579011"

 
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<partinfo>BBa_K4579011 short</partinfo>
 
<partinfo>BBa_K4579011 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>
  
This part contains code for a microcin which is an antimicrobial peptide targeting bacteria that are close relatives to the bacteria that natively produces them. These microcins are used in our project in an attempt to selectively kill the pathogen (this microcin will target Pantoea sp.).
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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.
<img src="https://static.igem.wiki/teams/4579/wiki/parts-collection-images/parts-collection-images/parts-collection-images/orangutan.jpeg" class= "center" width="300"
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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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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>.
===Usage and Biology===
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<h1>Categorization</h1>  
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4579011 SequenceAndFeatures</partinfo>
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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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<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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<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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===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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<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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<li><b>Inducible Microcin Expression Assemblies</b> – Assemblies of select microcins under the control of an inducible promoter system.</li>
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</ul>
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<h1>Usage and Biology</h1>
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This is a Type 3q part containing a putative, novel microcin (Mcc01) identified using the program <i>cinful</i> (Cole et al., 2022) which uses bioinformatics and alignment analysis to identify potential microcins from the inputted genomes of bacteria. We assembled this microcin into a constitutive expression assembly with a CP25 promoter (<html><a href="https://parts.igem.org/Part:BBa_K4579037">BBa_K4579037</a></html>) and <i>rpoC</i> terminator (<html><a href="https://parts.igem.org/Part:BBa_K4579036">BBa_K4579036</a></html>) to form the composite part <html><a href="https://parts.igem.org/Part:BBa_K4579039">BBa_K4579039</a></html>. This composite part plasmid was used in conjunction with secretion plasmid pSK01 (Kim et al., 2023) to engineer our chassis to secrete Mcc01 against <i>Pantoea spp.</i>
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==Composite Parts==
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<html><center><img src=https://static.igem.wiki/teams/4579/wiki/part-microcin.jpeg style="width:700px;height:auto;"></center></html>
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<center><b>Figure 2.</b> <i>The general schematic for our constitutive and inducible microcin assemblies with emphasis on the microcin part.</i></center>
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<ul>
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<li>Constitutive Mcc01 expression plasmid (<html><a href="https://parts.igem.org/Part:BBa_K4579039">BBa_K4579039</a></html>)</li>
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</ul>
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<h1>Design Notes</h1>
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We used <i>cinful</i> to predict and select potential microcins from genomes of bacteria related to our potential targets. To adapt our putative novel microcins to our cloning scheme, we first removed the native signal peptide sequence from the beginning of each microcin so that it could become fused with our system’s signal peptide (<html><a href="https://parts.igem.org/Part:BBa_K4579008">BBa_K4579008</a></html>) when expressed. Once the signal peptide sequence was removed, we obtained the original nucleotide sequence for each microcin by using the sequence start and stop positions provided in the <i>cinful</i> output to locate the sequence of the microcin within the genome. We then added flanking sequences to either side of each microcin that would add the necessary BsmBI and BsaI restriction sites for our cloning scheme (detailed in Figure 3 of our <html><a href="https://2023.igem.wiki/austin-utexas/parts">Parts</a></html> page). The final sequences of microcins - signal peptide + flanking restriction sites were then synthesized commercially as Gene Fragments by Twist Biosciences for use in our digestion-ligation reactions.
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<h1>Characterization</h1>
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All parts uploaded to the registry by our team, including this one, have been sequence confirmed.
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<h1>Source</h1>
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Mcc01 was identified from the genome of <i>Pantoea sp.</i> PSNIH3 using <i>cinful</i>.
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<h1>References</h1>
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<ol>
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<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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<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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<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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<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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<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>
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<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>
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</ol>
  
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===Functional Parameters===
 
<partinfo>BBa_K4579011 parameters</partinfo>
 
 
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<h1>Sequence and Features</h1>
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<partinfo>BBa_K4579011 SequenceAndFeatures</partinfo>

Latest revision as of 09:42, 12 October 2023


Mcc01 - Pantoea microcin 1

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 3q part containing a putative, novel microcin (Mcc01) identified using the program cinful (Cole et al., 2022) which uses bioinformatics and alignment analysis to identify potential microcins from the inputted genomes of bacteria. We assembled this microcin into a constitutive expression assembly with a CP25 promoter (BBa_K4579037) and rpoC terminator (BBa_K4579036) to form the composite part BBa_K4579039. This composite part plasmid was used in conjunction with secretion plasmid pSK01 (Kim et al., 2023) to engineer our chassis to secrete Mcc01 against Pantoea spp.

Composite Parts

Figure 2. The general schematic for our constitutive and inducible microcin assemblies with emphasis on the microcin part.

Design Notes

We used cinful to predict and select potential microcins from genomes of bacteria related to our potential targets. To adapt our putative novel microcins to our cloning scheme, we first removed the native signal peptide sequence from the beginning of each microcin so that it could become fused with our system’s signal peptide (BBa_K4579008) when expressed. Once the signal peptide sequence was removed, we obtained the original nucleotide sequence for each microcin by using the sequence start and stop positions provided in the cinful output to locate the sequence of the microcin within the genome. We then added flanking sequences to either side of each microcin that would add the necessary BsmBI and BsaI restriction sites for our cloning scheme (detailed in Figure 3 of our Parts page). The final sequences of microcins - signal peptide + flanking restriction sites were then synthesized commercially as Gene Fragments by Twist Biosciences for use in our digestion-ligation reactions.

Characterization

All parts uploaded to the registry by our team, including this one, have been sequence confirmed.

Source

Mcc01 was identified from the genome of Pantoea sp. PSNIH3 using cinful.

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]