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Abstract

Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.

Funding
This study was supported by the:
  • Wellcome Trust (Award WT103054MA)
    • Principle Award Recipient: DeanMadden
  • Wellcome Trust (Award WT103054MA)
    • Principle Award Recipient: JarosławBryk
  • FP7 People: Marie-Curie Actions (Award 300038)
    • Principle Award Recipient: JarosławBryk
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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/content/journal/acmi/10.1099/acmi.0.000596.v3
2023-09-06
2024-07-17
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References

  1. Endy D. Foundations for engineering biology. Nature 2005; 438:449–453 [View Article] [PubMed]
    [Google Scholar]
  2. Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat Methods 2013; 10:347–353 [View Article] [PubMed]
    [Google Scholar]
  3. Chen Y-J, Liu P, Nielsen AAK, Brophy JAN, Clancy K et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 2013; 10:659–664 [View Article] [PubMed]
    [Google Scholar]
  4. Lou C, Stanton B, Chen YJ, Munsky B, Voigt CA. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol 2012; 30:1137–1142 [View Article] [PubMed]
    [Google Scholar]
  5. Reeve B, Hargest T, Gilbert C, Ellis T. Predicting translation initiation rates for designing synthetic biology. Front Bioeng Biotechnol 2014; 2:1 [View Article] [PubMed]
    [Google Scholar]
  6. Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol 2019; 15:196–204 [View Article] [PubMed]
    [Google Scholar]
  7. Freemont PS. Synthetic biology industry: data-driven design is creating new opportunities in biotechnology. Emerg Top Life Sci 2019; 3:651–657 [View Article] [PubMed]
    [Google Scholar]
  8. Kelwick R, MacDonald JT, Webb AJ, Freemont P. Developments in the tools and methodologies of synthetic biology. Front Bioeng Biotechnol 2014; 2:60 [View Article] [PubMed]
    [Google Scholar]
  9. Young R, Haines M, Storch M, Freemont PS. Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly. Metab Eng 2021; 63:81–101 [View Article] [PubMed]
    [Google Scholar]
  10. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang R-Y et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010; 329:52–56 [View Article] [PubMed]
    [Google Scholar]
  11. Gleizer S, Ben-Nissan R, Bar-On YM, Antonovsky N, Noor E et al. Conversion of Escherichia coli to generate all biomass carbon from CO2. Cell 2019; 179:1255–1263 [View Article] [PubMed]
    [Google Scholar]
  12. Hoshika S, Leal NA, Kim MJ, Kim MS, Karalkar NB et al. Hachimoji DNA and RNA: a genetic system with eight building blocks. Science 2019; 363:884–887 [View Article] [PubMed]
    [Google Scholar]
  13. Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006; 440:940–943 [View Article] [PubMed]
    [Google Scholar]
  14. Schuler ML, Mantegazza O, Weber APM. Engineering C4 photosynthesis into C3 chassis in the synthetic biology age. Plant J 2016; 87:51–65 [View Article] [PubMed]
    [Google Scholar]
  15. Canton B, Labno A, Endy D. Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 2008; 26:787–793 [View Article] [PubMed]
    [Google Scholar]
  16. de Lorenzo V, Schmidt M. Biological standards for the knowledge-based bioeconomy: what is at stake. N Biotechnol 2018; 40:170–180 [View Article] [PubMed]
    [Google Scholar]
  17. Dixon J, Kuldell N. BioBuilding. In Methods of Enzymology vol 497 Elsevier; 2011 pp 255–271
    [Google Scholar]
  18. Penumetcha P, Lau K, Zhu X, Davis K, Eckdahl TT et al. Improving the lac system for synthetic biology. Bios 2010; 81:7–15 [View Article]
    [Google Scholar]
  19. Wolyniak MJ. Improved student linkage of Mendelian and molecular genetic concepts through a yeast-based laboratory module. Biochem Mol Biol Educ 2013; 41:163–172 [View Article] [PubMed]
    [Google Scholar]
  20. Bernstein R, Ingram K, Hart KM. BioBuilder: Synthetic Biology in the Lab O’Reilly Media, Inc; 2015
    [Google Scholar]
  21. Mitchell R, Dori YJ, Kuldell NH. Experiential engineering hrough iGEM—An undergraduate summer competition in synthetic biology. J Sci Educ Technol 2011; 20:156–160 [View Article]
    [Google Scholar]
  22. Freeman S, Eddy SL, McDonough M, Smith MK, Okoroafor N et al. Active learning increases student performance in science, engineering, and mathematics. Proc Natl Acad Sci 2014; 111:8410–8415 [View Article]
    [Google Scholar]
  23. Theobald EJ, Hill MJ, Tran E, Agrawal S, Arroyo EN et al. Active learning narrows achievement gaps for underrepresented students in undergraduate science, technology, engineering, and math. Proc Natl Acad Sci 2020; 117:6476–6483 [View Article] [PubMed]
    [Google Scholar]
  24. Gibson D. One-step enzymatic assembly of DNA molecules up to several hundred kilobases in size. Protoc Exch 2009 [View Article]
    [Google Scholar]
  25. Gibson DG. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 2011; 498:349–361 [View Article]
    [Google Scholar]
  26. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods 2013; 10:354–360 [View Article] [PubMed]
    [Google Scholar]
  27. Cox RS, Surette MG, Elowitz MB. Programming gene expression with combinatorial promoters. Mol Syst Biol 2007; 3:145 [View Article] [PubMed]
    [Google Scholar]
  28. Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 1996; 6:178–182 [View Article] [PubMed]
    [Google Scholar]
  29. Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci 2000; 97:11984–11989 [View Article] [PubMed]
    [Google Scholar]
  30. Buhk HJ. Synthetic biology and its regulation in the European Union. N Biotechnol 2014; 31:528–531 [View Article] [PubMed]
    [Google Scholar]
  31. Hansson SO. How to be cautious but open to learning: time to update biotechnology and GMO legislation. Risk Anal 2016; 36:1513–1517 [View Article] [PubMed]
    [Google Scholar]
  32. Reeves RG, Denton JA, Santucci F, Bryk J, Reed FA. Scientific standards and the regulation of genetically modified insects. PLoS Negl Trop Dis 2012; 6:e1502 [View Article] [PubMed]
    [Google Scholar]
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