pDGB1 omega BACKBONE for Bacterial GoldenBraid Cloning

This backbone combined with the proper insert BBa_K3505041 results in the comlpete Vectors BBa_K3505010 BBa_K3505011

Fig.1: Ω2 plasmid Features and Restiction Sites.

Fig.2: Ω1R 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 Ω1R BBa_K3505036 BBa_K3505037

pDGB1 Ω1R and Ω2 verification with no insert

Fig.3: pDGB1 omega1R Digested with BamHI. Expected bands 3153, 382, 239

Fig.4: U3 C3 pDGB1 omega2 Digested with EcoRV and HidIII. Expected bands 2900, 398, 223

Source

Christos Batianis[3]

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 931
Illegal NgoMIV site found at 1333
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
[3Damalas, S., Batianis, C., Martin‐Pascual, M., Lorenzo, V. and Martins dos Santos, 2020. SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial