Genetic constructs creation using Golden Gate cloning method

  • O. I. Varchenko
  • B. M. Krasyuk
  • A. A. Fedchunov
  • O. V. Zimina
  • M. F. Parii
  • Yu. V. Symonenko

Abstract

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

References

Redberry G. Gene silencing: new research. New York: Nova Science Publishers. 2006. 212 p.

Kahl L.J., Endy D. A survey of enabling technologies in synthetic biology. J. Biol. Eng. 2013. Vol. 7. P. 13.

Anderson J.C., Dueber J.E., Leguia M., Wu G.C., Goler J.A., Arkin A.P., Keasling J.D. BglBricks: A flexible standard for biological part assembly. J. Biol. Eng. 2010. Vol. 4. P. 1.

Shetty R.P., Endy D., Knight T.F. Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2008. Vol. 2. P. 5.

Shetty R., Lizarazo M., Rettberg R., Knight T.F. Assembly of BioBrick standard biological parts using three antibiotic assembly. Methods Enzymol. 2011. Vol. 498. P. 311−326.

Sleight S.C., Bartley B.A., Lieviant J.A., Sauro H.M. In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res. 2010. Vol. 38. P. 2624−2636.

Xu P., Vansiri A., Bhan N., Koffas M.A.G. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli. ACS Synth. Biol. 2012. Vol. 1. P. 256−266.

Engler C., Kandzia R., Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008. Vol. 3. P. 3647.

Ellis T., Adie T., Baldwin G.S. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr. Biol. (Camb). 2011. Vol. 3. P. 109−118.

Weber E., Engler C., Gruetzner R., Werner S., Marillonnet, S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011. Vol. 6. P. 16765.

Sarrion-Perdigones A., Falconi E.E., Zandalinas S.I., Juárez P., Fernándezdel-Carmen A., Granell A., Orzaez D. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One. 2011. Vol. 6. P. 21622.

Werner S., Engler C., Weber E., Gruetzner R., Marillonnet S. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng. Bugs. 2012. Vol. 3. P. 38−43.

Engler C., Gruetzner R., Kandzia R., Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PloS One. 2009. Vol. 4, № 5. P. 5553.

Chalfie M., Tu Y., Euskirchen G., Ward W.W., Prasher D.C. Green Fluorescent Protein as a Marker for Gene Expression. Science. 1994. Vol. 263. P. 802–805.

Chiu W-L., Niwa Y., Zeng W., Hirano T., Kobayashi H., Sheen J., Engineered GFP as a vital reporter in plants. Curr. Biol. 1996. Vol. 6. P. 325–330.

Ow D., Jacobs J., Howell S. Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc. Natl. Acad. Sci. U.S.A. 1987. Vol. 84. P. 4870–4874.

Guilley H., Dudley R., Jonard G., Balàzs E., Richards, K., Transcription of Cauliflower mosaic virus DNA: detection of pro-moter sequences, and characterization of transcripts. Cell. 1982. Vol. 30. P. 763–773.

Kay R., Chan A., Daly M., McPherson J. Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes. Science. 1987. Vol. 236. P. 1299–1302.

Dedonder A., Rethy R., Fredericq H., Van Montagu M., Krebbers E. Arabidopsis rbcS genes are differentially regulated by light. Plant Physiol. 1993. Vol. 101. P. 801–808.

McGrath J., Terzaghi W., Sridhar P., Cashmore A., Pichersky E. Sequence of the fourth and fifth Photosystem II type I chloro-phyll a/b-binding protein genes of Arabidopsis thaliana and evidence for the presence of a full complement of the extended CAB gene family. Plant Mol. Biol. 1992. Vol. 19. P. 725–733.

Gallie D.R., Sleat D.E., Watts J.W., Turner P.C., Wilson T.M. The 5’-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987. Vol. 15. P. 3257–3273.

Making Calcium Competent Cells. URL: http://mcb.berkeley.edu/labs/krantz/protocols/calcium_comp_cells.pdf (Last ac-cessed: 5.04.2019).

Froger A., Hall J.E. Transformation of plasmid DNA into E. coli using the heat shock method. Journal of visualized experi-ments: JoVE. 2007. Vol. 6.

Lerner C.G., Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic acids research. 1990. Vol. 18, № 15. P. 4631.

Lezin G., Kosaka Y., Yost H.J., Kuehn M.R., Brunelli L. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles. PLoS One. 2011. Vol. 6, № 8. P. 23457.

URL: http://sites.bio.indiana.edu/~pikaardlab/PDFs%20and%20protocol%20files%20/agrotransform.html (Last accessed: 1.04.2019).

Bendandi M., Marillonnet S., Kandzia R., Thieme F., Nickstadt A., Herz S., Frode R., Inoges S., Lopez-Diaz de Cerio A., Soria, E., Villanueva H., Vancanneyt G., McCormick A., Tuse D., Lenz J., Butler-Ransohoff J.E., Klimyuk V., Gleba Y. Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin’s lymphoma. Ann. Oncol. 2010. Vol. 21. P. 2420–2427.

Tuse, D. et al. Clinical Safety and Immunogenicity of Tumor-Targeted, Plant-Made Id-KLH Conjugate Vaccines for Follicular Lymphoma. BioMed. Res. Int. 2015. P. 648143.

Werner S., Breus O., Symonenko Y., Marillonnet S., Gleba Y. High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc Natl Acad Sci USA. 2011. Vol. 108, № 34. P. 14061–14066.

Hahn S., Giritch A., Bartels D., Bortesi L., Gleba Y. A novel and fully scalable Agrobacterium spray-based process for manu-facturing cellulases and other costsensitive proteins in plants. Plant Biotechnol J. 2015. Vol. 13, № 5. P. 708–716.

Schulz S., Stephan A., Hahn S., Bortesi L., Jarczowski F., Bettmann U. et al. Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins. Proc Natl Acad Sci USA. 2015. Vol. 112. P. E5454–5460.