Difference between revisions of "Part:BBa K5119080"

 
 
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Through our research, we offer a collection of parts <b>(</b>  <a href="https://parts.igem.org/Part:BBa_K5119000">BBa_K5119000</a>to  <a href="https://parts.igem.org/Part:BBa_K5119089">BBa_K5119089</a><b>)</b> that enables researchers to assemble their own plasmid that can replicate in both gram-positive species and <i>E. coli</i>, with the added functionality of secreting enzymes capable of degrading gliadin. Explore the entire collection of parts associated with UT Austin's 2024 iGEM project on the <a href="https://2024.igem.wiki/austin-utexas/parts">Parts webpage.</a>
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<h1>Introduction</h1> 
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<img src=https://static.igem.wiki/teams/5119/parts-collection/igem-team-logo.png style="float: right; width:300px; height: auto;"> 
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About 1% of the world population is affected by celiac disease, <sup>[3]</sup> an autoimmune disorder triggered by the ingestion of gluten, a protein commonly found in wheat, barley, and rye.<sup>[4]</sup> This immune response can cause significant intestinal damage from chronic inflammation, nutrient malabsorption, and even lactose intolerance, making it crucial to find effective treatments. This is further underscored by the widespread presence of gluten in the human diet. The UT Austin 2024 iGEM team seeks to alleviate the burden of celiac disease by developing a collection of parts capable of secreting proteases in a bacterium specifically designed to degrade gliadin, the primary immunogenic component of gluten.<sup>[5]</sup> By engineering this bacterium to break down gliadin in a sustained and localized manner, the team aims to prevent the harmful effects of accidental gluten ingestion, offering a solution to improve the lives of individuals with celiac disease. For more details, please visit our <a href="https://2024.igem.wiki/austin-utexas/description">Project Description.</a>
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<center><img src=https://static.igem.wiki/teams/5119/parts-collection/comppartsoverview.jpg style="width:700px; height: auto;"></center> 
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<b>Figure 1:</b> The UT-Austin 2024 iGEM parts collection. This collection includes twenty-two constitutive antibiotic resistance promoters & RBS (Type 2), nine secretion tags (Type 3a), two reporter proteins and four reporter proteins & enzymes (Type 3b), a rpoC terminator (Type 4), and three plasmid backbones (Type 56781). Created with <i>Biorender.com.</i>     
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<br></br>
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Our parts collection consists of a diverse array of plasmid backbones (Type 56781), promoters & RBS (Type 2), signal peptides (Type 3a), and enzyme coding sequences (Type 3b), designed to enable the modular engineering of plasmids that express gliadin-degrading enzymes. Drawing from the methodologies established in the Yeast Toolkit<sup>[6]</sup> and the Bee Microbiome Toolkit,<sup>[2]</sup> our collection allows for the seamless arrangement of genetic parts using type IIS enzymatic Golden Gate Assembly (GGA). Similar to the BTK, our plasmid elements - including broad-host-range promoters, coding sequences, and antibiotic resistance genes - can be independently replaced to optimize performance for specific bacterial hosts. The Ribosome Binding Site (RBS) for all promoters were native to the original antibiotic resistance gene. For all Type 2 parts, the RBS site is included in the individual promoter sequences.
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<center><img src=https://static.igem.wiki/teams/5119/parts-collection/genplasmidstructure.jpg style="width:800px; height: auto;"></center> 
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<b> Figure 2:</b> An example of an assembly plasmid containing five part types: a plasmid backbone (Type 56781), a promoter (Type 2), a secretion tag (Type 3a), an enzyme coding region (Type 3b), and a terminator (Type 4). Part Type numbers and overhangs are derived from the Yeast Toolkit<sup>[6]</sup>  and the Bee Microbiome Toolkit<sup>[2]</sup>  and follow their guidelines. Created with <i>Biorender.com.</i>
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<br></br>
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Our research focuses on four key areas: 
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<ul>
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  <li><b>Shuttle plasmid backbones in gram-positive bacteria</b></li>
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  <li><b>Weakly constitutive promoters from antibiotic resistance genes</b></li>
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  <li><b>Gliadin-degrading enzyme expression </b></li>
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  <li><b>Protein secretion using SecII-dependent signal tags </b></li>
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</ul>
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<p>The parts in our collection work synergistically to achieve varying levels of constitutive production and efficient protein secretion. To investigate this, we created numerous composite parts to identify optimal promoters and secretion tags, focusing on their transcriptional strength and secretion efficiency. These constructs were then inserted into three domesticated backbones, designed to serve as modular plasmid vectors for ideal functionality.</p>
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<h1>Categorization</h1> 
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<h3>Basic parts</h3>
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<ul>
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  <li><b>Promoters (Type 2)</b> - 22 broad-host-range promoters were selected from common antibiotic resistance gene cassettes used in engineered plasmids. Each promoter was tested for its relative strengths with a red fluorescent protein in a pIB184 backbone.</li>
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  <li><b>Coding Sequences (Type 3a + 3b)</b>
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<ul>
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<li><b>Signal tags (3a)</b> – Nine Sec-dependent signal tags, previously tested in <i>E. coli</i> or derived from gram-positive bacteria, were paired with fluorescent proteins and tested for secretion efficiency. They were further evaluated with gliadin-degrading enzymes.</li>
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<li><b>Proteins & Proteases (3b)</b> – Fluorescent proteins such as mScarlet and sfGFP were used as reporters to assess protein secretion. Well-characterized gliadin-degrading enzymes like Kuma030 and AN-PEP were tested for their activity. </li>
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  </ul>
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</li>
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  <li><b>Backbone (Type 56781)</b> – An <i>E. coli</i> expression plasmid and two shuttle vector plasmids with origins that replicate in both <i>E. coli</i> and gram-positive bacteria were modified to create compatible plasmid backbones. They were paired with a green fluorescent protein, signal tags, and gliadin-degrading enzymes.</li>
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</ul>
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<h3>Composite parts</h3>
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<b>Composite secretion plasmids</b> – These plasmids were created to assess the efficiency of using different tags to secrete reporter proteins or gliadin-degrading enzymes from bacteria. 
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<b>Composite promoter plasmids</b> – These plasmids were designed to assess the transcriptional strength of the various promoters through fluorescence tests using the iGEM Measurement Kit containing calibration beads for plate readers.
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<h1>Part Design and Construction</h1>
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All composite plasmids have the same rpoC terminator: <a href="https://parts.igem.org/Part:BBa_K5119034">BBa_K5119034</a> 
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The PelB secretion tag and the mScarlet reporter protein sequence were inserted into the pET28a backbone that contains a T7 promoter. This assembly plasmid was made with the BsaI NEB Golden Gate Assembly Kit.<sup>[9]</sup> The purpose of this part was to test the ability PelB to secrete mScarlet protein from <i>E. coli</i> Tuner DE3 cells, under the control of an inducible T7 promoter. We used a microplate reader to measure the fluorescence in the supernatant, which allowed for the quantification of secretion efficiency.
  
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<span class='h3bb'>Sequence and Features</span>
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<h1>Characterization</h1>
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<center><img src=https://static.igem.wiki/teams/5119/secretion-results/secretion-results-figure-4.png style="width:900px; height: auto;"></center>  
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<b>Figure 3. Secretion of mScarlet when paired with secretion tags in pET28a.</b> Secretion efficiencies and amount of fluorescence generated in the extracellular milieu relative to the number of cells across several trials of induction. <b>A)</b> Supernatant fluorescence over culture fluorescence <b>B)</b> Supernatant fluorescence over OD of culture. Data for each signal tags averaged out across 4 trials for each induction group. Fluorescence measured at one timepoint from a micro plate reader using cultures grown for 20 hours of incubation at 37 °C. Supernatant fluorescence taken from supernatant of cells spun down at 7000 rpm at 4°C for 10 mins.
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<center><img src=https://static.igem.wiki/teams/5119/secretion-results/secretion-results-figure-7.png style="width:900px; height: auto;"></center> 
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<b>Figure 3. Secretion of mScarlet when paired with secretion tags in pET28a under the control of pLysS. a)</b> Supernatant fluorescence over culture fluorescence for pBTK1030 + signal tags <b>b)</b> Supernatant fluorescence over culture OD660 for pBTK1030 + signal tags. <b>c)</b> Supernatant fluorescence over culture fluorescence for Kan full length promoter (Kan FL) and variants + signal tags <b>d)</b> Supernatant fluorescence over culture OD660 for Kan full length promoter (Kan FL) and variants + signal tags. Results were averaged across 3 trials for each signal tag group and normalized for LB media background fluorescence. Fluorescence measure taken at one timepoint from a micro plate reader after 20 hours of incubation at 37 °C. Supernatant fluorescence readings taken from supernatant of cultures spun down at 7000 rpm at 4°C for 10 mins.
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The results for PelB-tagged constructs had variable secretion efficiency in pET28a in Tuner DE3, however showed more consistent data in pIB184 and pET28a under control of the pLys plasmid from BL21 DE3. In the pET28a plasmid, one of the challenges encountered was the high transcription rate of the T7 promoter, which led to instability and inconsistent secretion. Although fluorescence was observed in the supernatant, suggesting some level of secretion, the efficiency was not significantly higher than the controls. The instability of the pET28a plasmid due to the T7 promoter’s high transcription rate appeared to affect the performance of the PelB signal peptide. Additionally, there were signs of cell lysis in some of our assays, which could have contributed to the observed fluorescence in the supernatant, complicating the interpretation of the data. PelB was one of the better performing signal peptides for secretion in <i>E. coli</i>, and may provide more significant data in gram-positives such as <i>L. lactis</i>.
  
  
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===Functional Parameters===
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<b>Figure 4:</b> List of Composite Promoter plasmids and Composite secretion plasmids. Basic parts of the same type can be interchanged. The table provides definitions for part symbols in SBOL language. Created with <i>Biorender.com.</i>
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<h1>Sequence and Features</h1> 
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<partinfo>BBa_K5119080 SequenceAndFeatures</partinfo>
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<br>
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<h1>References</h1>
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<ol>
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  <li> Novick, R. P., & Murphy, E. (1985). MLS-resistance determinants in Staphylococcus aureus and their molecular evolution. Journal of Antimicrobial Chemotherapy, 16(suppl A), 101–110. https://doi.org/10.1093/jac/16.suppl_a.101 </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, A. D., 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. https://doi.org/10.1021/acssynbio.7b00399 </li>
 +
<li> Lebwohl, B., Sanders, D. S., & Green, P. H. R. (2018). Coeliac disease. Lancet (London, England), 391(10115), 70–81. https://doi.org/10.1016/S0140-6736(17)31796-8</li>
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<li> Celiac Disease Foundation. (2024). What Is Celiac Disease? Celiac Disease Foundation; Celiac Disease Foundation. https://celiac.org/about-celiac-disease/what-is-celiac-disease/</li>
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<li> Barone, M.V., Troncone, R., Auricchio, S. Gliadin Peptides as Triggers of the Proliferative and Stress/Innate Immune Response of the Celiac Small Intestinal Mucosa. Int. J. Mol. Sci. 2014, 15, 20518-20537. https://doi.org/10.3390/ijms151120518 </li>
 +
<li> 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. https://doi.org/10.1021/sb500366v </li>
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<li>LaFleur, T.L., Hossain, A. & Salis, H.M. Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nat Commun 13, 5159 (2022). https://doi.org/10.1038/s41467-022-32829-5 </li>
 +
<li> Engler C., Kandzia R., Marillonnet S. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE 3(11): e3647. https://doi.org/10.1371/journal.pone.0003647 </li>
 +
<li> Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Evans, T. C., & Lohman, G. J. S. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology, 7(11), 2665–2674. https://doi.org/10.1021/acssynbio.8b00333 </li>
 +
<li> Sambrook, J., & Russel, D. W. (2001). Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press. </li>
 +
<li> Baldwin, G., Haddock-Angelli, T., Beal, J., Dwijayanti, A., Storch, M., Farny, N., Telmer, C., Vignoni, A., Tennant, R., & Rutten, P. (2019). Calibration Protocol - Plate Reader Fluorescence Calibration v3. ACS Synthetic Biology. https://doi.org/10.17504/protocols.io.6zrhf56 </li>
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<li> Beal, J., Haddock-Angelli, T., Gershater, M., Sanchania, V., Buckley-Taylor, R., Baldwin, G., Farny, N., Tennant, R., & Rutten, P. (2020). Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles v4. iGE. https://dx.doi.org/10.17504/protocols.io.bht7j6rn </li>
 +
<li> Biswas, I., Jha, J. K., & Fromm, N. (2008). Shuttle expression plasmids for genetic studies in Streptococcus
 +
mutans. Microbiology (Reading, England), 154(Pt 8), 2275–2282. https://doi.org/10.1099/mic.0.2008/019265-0 </li>
 +
<li>Samperio, S., Guzmán-Herrador, D. L., May-Cuz, R., Martín, M. C., Álvarez, M. A., & Llosa, M. (2021, January 22). Conjugative DNA transfer from E. coli to transformation-resistant lactobacilli. Frontiers. https://doi.org/10.3389/fmicb.2021.606629 </li>
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<li> Jennings, M. P., & Beacham, I. R. (1990). Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins. Journal of bacteriology, 172(3), 1491–1498. https://doi.org/10.1128/jb.172.3.1491-1498.1990 </li>
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<li>Ahmadi, Z., Farajnia, S., Farajzadeh, D., Pouladi, N., Pourvatan, N., Karbalaeimahdi, M., Shayegh, F., & Arya, M. (2023). Optimized Signal Peptide for Secretory Expression of Human Recombinant Somatropin in E. coli. Advanced pharmaceutical bulletin, 13(2), 339–349. https://doi.org/10.34172/apb.2023.037 </li>
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<li>Benson, S. A., & Silhavy, T. J. (1983). Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12. Cell, 32(4), 1325–1335. https://doi.org/10.1016/0092-8674(83)90313-6 </li>
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<li>Bowers, C. W., Lau, F., & Silhavy, T. J. (2003). Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli. Journal of bacteriology, 185(19), 5697–5705. https://doi.org/10.1128/JB.185.19.5697-5705.2003 </li>
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<li>Takahara, M., Hibler, D. W., Barr, P. J., Gerlt, J. A., & Inouye, M. (1985). The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli. Journal of Biological Chemistry, 260(5), 2670–2674. https://doi.org/10.1016/S0021-9258(18)89413-3 </li>
 +
<li>Li, Z., Leung, W., Yon, A., Nguyen, J., Perez, V. C., Vu, J., Giang, W., Luong, L. T., Phan, T., Salazar, K. A., Gomez, S. R., Au, C., Xiang, F., Thomas, D. W., Franz, A. H., Lin-Cereghino, J., & Lin-Cereghino, G. P. (2010). Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris. Protein expression and purification, 72(1), 113–124. https://doi.org/10.1016/j.pep.2010.03.004 </li>
 +
<li>Pechsrichuang, P., Songsiriritthigul, C., Haltrich, D., Roytrakul, S., Namvijtr, P., Bonaparte, N., & Yamabhai, M. (2016). OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, 5(1), 1200. https://doi.org/10.1186/s40064-016-2893-y </li>       
 +
<li>Power, B. E., Ivancic, N., Harley, V. R., Webster, R. G., Kortt, A. A., Irving, R. A., & Hudson, P. J. (1992). High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal. Gene, 113(1), 95–99. https://doi.org/10.1016/0378-1119(92)90674-E </li>
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<li>de Cock, H., Overeem, W., Tommassen, J. (1992). Biogenesis of outer membrane protein PhoE of Escherichia coli: Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein. Journal of Molecular Biology, 224(2), 369-379. https://www.sciencedirect.com/science/article/pii/0022283692910016?pes=vor </li>
 +
<li>Adams, H., Scotti, P. A., De Cock, H., Luirink, J., & Tommassen, J. (2002). The presence of a helix breaker in the hydrophobic core of signal sequences of secretory proteins prevents recognition by the signal-recognition particle in Escherichia coli. European journal of biochemistry, 269(22), 5564–5571. https://doi.org/10.1046/j.1432-1033.2002.03262.x </li>
 +
<li>Ravn, P., Arnau, J., Madsen, S. M., Vrang, A., & Israelsen, H. (2000). The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis. Gene, 242(1-2), 347–356. https://doi.org/10.1016/s0378-1119(99)00530-2 </li>
 +
<li>Ravn, P., Arnau, J., Madsen, S. M., Vrang, A., & Israelsen, H. (2003). Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis. Microbiology (Reading, England), 149(Pt 8), 2193–2201. https://doi.org/10.1099/mic.0.26299-0 </li>
 +
<li>Morello, E., Bermúdez-Humarán, L. G., Llull, D., Solé, V., Miraglio, N., Langella, P., & Poquet, I. (2008). Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. Journal of molecular microbiology and biotechnology, 14(1-3), 48–58. https://doi.org/10.1159/000106082 </li>
 +
<li>Ng, D. T., & Sarkar, C. A. (2013). Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Applied and environmental microbiology, 79(1), 347–356. https://doi.org/10.1128/AEM.02667-12</li>                 
 +
</ol>
 +
<br>

Latest revision as of 12:05, 2 October 2024


pET28a + mScarlet + pelB Secretion plasmid

Through our research, we offer a collection of parts ( BBa_K5119000to BBa_K5119089) that enables researchers to assemble their own plasmid that can replicate in both gram-positive species and E. coli, with the added functionality of secreting enzymes capable of degrading gliadin. Explore the entire collection of parts associated with UT Austin's 2024 iGEM project on the Parts webpage.

Introduction

About 1% of the world population is affected by celiac disease, [3] an autoimmune disorder triggered by the ingestion of gluten, a protein commonly found in wheat, barley, and rye.[4] This immune response can cause significant intestinal damage from chronic inflammation, nutrient malabsorption, and even lactose intolerance, making it crucial to find effective treatments. This is further underscored by the widespread presence of gluten in the human diet. The UT Austin 2024 iGEM team seeks to alleviate the burden of celiac disease by developing a collection of parts capable of secreting proteases in a bacterium specifically designed to degrade gliadin, the primary immunogenic component of gluten.[5] By engineering this bacterium to break down gliadin in a sustained and localized manner, the team aims to prevent the harmful effects of accidental gluten ingestion, offering a solution to improve the lives of individuals with celiac disease. For more details, please visit our Project Description.
Figure 1: The UT-Austin 2024 iGEM parts collection. This collection includes twenty-two constitutive antibiotic resistance promoters & RBS (Type 2), nine secretion tags (Type 3a), two reporter proteins and four reporter proteins & enzymes (Type 3b), a rpoC terminator (Type 4), and three plasmid backbones (Type 56781). Created with Biorender.com.

Our parts collection consists of a diverse array of plasmid backbones (Type 56781), promoters & RBS (Type 2), signal peptides (Type 3a), and enzyme coding sequences (Type 3b), designed to enable the modular engineering of plasmids that express gliadin-degrading enzymes. Drawing from the methodologies established in the Yeast Toolkit[6] and the Bee Microbiome Toolkit,[2] our collection allows for the seamless arrangement of genetic parts using type IIS enzymatic Golden Gate Assembly (GGA). Similar to the BTK, our plasmid elements - including broad-host-range promoters, coding sequences, and antibiotic resistance genes - can be independently replaced to optimize performance for specific bacterial hosts. The Ribosome Binding Site (RBS) for all promoters were native to the original antibiotic resistance gene. For all Type 2 parts, the RBS site is included in the individual promoter sequences.
Figure 2: An example of an assembly plasmid containing five part types: a plasmid backbone (Type 56781), a promoter (Type 2), a secretion tag (Type 3a), an enzyme coding region (Type 3b), and a terminator (Type 4). Part Type numbers and overhangs are derived from the Yeast Toolkit[6] and the Bee Microbiome Toolkit[2] and follow their guidelines. Created with Biorender.com.

Our research focuses on four key areas:
  • Shuttle plasmid backbones in gram-positive bacteria
  • Weakly constitutive promoters from antibiotic resistance genes
  • Gliadin-degrading enzyme expression
  • Protein secretion using SecII-dependent signal tags

The parts in our collection work synergistically to achieve varying levels of constitutive production and efficient protein secretion. To investigate this, we created numerous composite parts to identify optimal promoters and secretion tags, focusing on their transcriptional strength and secretion efficiency. These constructs were then inserted into three domesticated backbones, designed to serve as modular plasmid vectors for ideal functionality.

Categorization

Basic parts

  • Promoters (Type 2) - 22 broad-host-range promoters were selected from common antibiotic resistance gene cassettes used in engineered plasmids. Each promoter was tested for its relative strengths with a red fluorescent protein in a pIB184 backbone.
  • Coding Sequences (Type 3a + 3b)
    • Signal tags (3a) – Nine Sec-dependent signal tags, previously tested in E. coli or derived from gram-positive bacteria, were paired with fluorescent proteins and tested for secretion efficiency. They were further evaluated with gliadin-degrading enzymes.
    • Proteins & Proteases (3b) – Fluorescent proteins such as mScarlet and sfGFP were used as reporters to assess protein secretion. Well-characterized gliadin-degrading enzymes like Kuma030 and AN-PEP were tested for their activity.
  • Backbone (Type 56781) – An E. coli expression plasmid and two shuttle vector plasmids with origins that replicate in both E. coli and gram-positive bacteria were modified to create compatible plasmid backbones. They were paired with a green fluorescent protein, signal tags, and gliadin-degrading enzymes.

Composite parts

Composite secretion plasmids – These plasmids were created to assess the efficiency of using different tags to secrete reporter proteins or gliadin-degrading enzymes from bacteria.
Composite promoter plasmids – These plasmids were designed to assess the transcriptional strength of the various promoters through fluorescence tests using the iGEM Measurement Kit containing calibration beads for plate readers.

Part Design and Construction

All composite plasmids have the same rpoC terminator: BBa_K5119034
The PelB secretion tag and the mScarlet reporter protein sequence were inserted into the pET28a backbone that contains a T7 promoter. This assembly plasmid was made with the BsaI NEB Golden Gate Assembly Kit.[9] The purpose of this part was to test the ability PelB to secrete mScarlet protein from E. coli Tuner DE3 cells, under the control of an inducible T7 promoter. We used a microplate reader to measure the fluorescence in the supernatant, which allowed for the quantification of secretion efficiency.

Characterization



Figure 3. Secretion of mScarlet when paired with secretion tags in pET28a. Secretion efficiencies and amount of fluorescence generated in the extracellular milieu relative to the number of cells across several trials of induction. A) Supernatant fluorescence over culture fluorescence B) Supernatant fluorescence over OD of culture. Data for each signal tags averaged out across 4 trials for each induction group. Fluorescence measured at one timepoint from a micro plate reader using cultures grown for 20 hours of incubation at 37 °C. Supernatant fluorescence taken from supernatant of cells spun down at 7000 rpm at 4°C for 10 mins.

Figure 3. Secretion of mScarlet when paired with secretion tags in pET28a under the control of pLysS. a) Supernatant fluorescence over culture fluorescence for pBTK1030 + signal tags b) Supernatant fluorescence over culture OD660 for pBTK1030 + signal tags. c) Supernatant fluorescence over culture fluorescence for Kan full length promoter (Kan FL) and variants + signal tags d) Supernatant fluorescence over culture OD660 for Kan full length promoter (Kan FL) and variants + signal tags. Results were averaged across 3 trials for each signal tag group and normalized for LB media background fluorescence. Fluorescence measure taken at one timepoint from a micro plate reader after 20 hours of incubation at 37 °C. Supernatant fluorescence readings taken from supernatant of cultures spun down at 7000 rpm at 4°C for 10 mins.
The results for PelB-tagged constructs had variable secretion efficiency in pET28a in Tuner DE3, however showed more consistent data in pIB184 and pET28a under control of the pLys plasmid from BL21 DE3. In the pET28a plasmid, one of the challenges encountered was the high transcription rate of the T7 promoter, which led to instability and inconsistent secretion. Although fluorescence was observed in the supernatant, suggesting some level of secretion, the efficiency was not significantly higher than the controls. The instability of the pET28a plasmid due to the T7 promoter’s high transcription rate appeared to affect the performance of the PelB signal peptide. Additionally, there were signs of cell lysis in some of our assays, which could have contributed to the observed fluorescence in the supernatant, complicating the interpretation of the data. PelB was one of the better performing signal peptides for secretion in E. coli, and may provide more significant data in gram-positives such as L. lactis.

Figure 4: List of Composite Promoter plasmids and Composite secretion plasmids. Basic parts of the same type can be interchanged. The table provides definitions for part symbols in SBOL language. Created with Biorender.com.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 5110
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5040
  • 23
    INCOMPATIBLE WITH RFC[23]
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    Illegal NgoMIV site found at 217
    Illegal NgoMIV site found at 3260
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    Illegal NgoMIV site found at 5008
  • 1000
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