Part:BBa_K3505040

pDGB1 alpha BACKBONE for Bacterial GoldenBraid Cloning

This backbone combined with the proper insert BBa_K3505041 results in the comlpete Vectors BBa_K3505008BBa_K3505009

Fig.1: a1R plasmid Features and Restiction Sites.

Fig.1: a2 plasmid Features and Restiction Sites.

Usage and Biology

This Parts Collection contains 5 plasmid vectors for GoldenBraid TypeIIS cloning in bacteria, by exploiting the Standardized European Vector Architecture (SEVA) plasmid collection.

GoldenBraid (GB) is a DNA assembly strategy for Plant Synthetic Biology based on Type IIS enzymes[1]. We modified two SEVA plasmids, pSEVA23 and pSEVA43[2], to create two α (alpha) and two Ω (omega) destination vectors, which allow multipartite and multigenic modular assembly in forward or reverse orientation. This supports fully customized design of complex multi-TU constructs, which we needed in our project. Notably, we have designed the vectors so the antibiotic resistance gene and ORI is interchangeable, so any can be chosen from the huge variety of the SEVA collection, making our Parts Collection universal.

Moreover, in this collection we added the Universal Part Domesticator plasmid (pUPD2), an pSB1C3-based vector that allows entry/domestication of Level 0 parts into the GoldeBraid standard.

With this collection, iGEM teams can combine the power of modular cloning and the standardized nature of SEVA plasmids to build and test huge constructs in several prokaryotic chassis.

Experimental Use and Experience

The following parts were cloned in these 2 vectors BBa_K3505025 BBa_K3505026 BBa_K3505027 BBa_K3505028 BBa_K3505029 BBa_K3505030 BBa_K3505031 BBa_K3505032 BBa_K3505033 BBa_K3505034 BBa_K3505035 BBa_K3505038 BBa_K3505039

pDGB1 a1R an a2 verification with no insert

Fig.3: pDGB1 a1R Digested with BamHI. Expected bands 2847, 385, 239 and pDGB1 a2 Digested with HindIII. Expected bands 2847, 416, 206

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1274
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

• [1]Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). “GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology.” Plant Physiology , 162 (3) 1618-1631; DOI: 10.1104/pp.113.217661
• [2]Esteban Martínez-García, Angel Goñi-Moreno, Bryan Bartley, James McLaughlin, Lucas Sánchez-Sampedro, Héctor Pascual del Pozo, Clara Prieto Hernández, Ada Serena Marletta, Davide De Lucrezia, Guzmán Sánchez-Fernández, Sofía Fraile, Víctor de Lorenzo, SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts, Nucleic Acids Research, Volume 48, Issue D1, 08 January 2020, Pages D1164–D1170, https://doi.org/10.1093/nar/gkz1024