Plasmid

Part:BBa_K3505008

Designed by: Venetios Michelioudakis   Group: iGEM20_Thessaly   (2020-10-16)
Revision as of 11:57, 23 October 2020 by Venetios (Talk | contribs) (→‎Experimental Use and Experience)


pDGB1 alpha 1R (α1R) for Bacterial GoldenBraid.

Level a vector for Golden Braid Cloning. Having cargo LacZa for blue white screening between BsaI and BsmBI sites.Resistance in Kanamycin and ori pBBR1 for multiple protein expression capacities.


Fig.1: a1R plasmid Features and Restiction Sites.
Fig.2: a1R Map.

Usage and Biology

Golden Braid (GB) is a DNA assembly strategy for Plant Synthetic Biology based on Type IIS enzymes. It is also compatible for MoClo assembly. The sequences must not contain BsmBI and BsaI sites! Domestication may be done in order to vanish BsaI and BsmBI sites from the inner sequence.

GB proposes an alternative view of modular cloning, and essentially the change is that you can infinitely assemble new vectors by performing “braids”[1]. Using BsmBI another big advantage is the use of a single level 0 vector (pUPD and pUPD2, where pUPD2BBa_K3505007is derived from iGEM-borne pSB1C3) for any GB part one needs. Then combining the desirable fragments from level 0; ‘level alpha’ (level a) cloning is succeeded creating Transcription Units (TU). Desirable TUs are combined to result in (Level Ω) cloning. • Using BsmBI for Level 0 modules and ‘level omega’ (Level Ω) • Using BsaI for ‘Le ting vel alpha’ (Level a)

But this is not the end, Golden Braid outperforms Golden Gate (which everyone knows) because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression of Golden Gate. Binary assembly of 2 Level Ω (Level 2 for MoClo) result in an alpha vector. Again Binary assembly of 2 alpha result in an omega vector. With this assembly you can insert step by step as many parts and as many TUs you want with high efficiency. We constructed level α and level Ω plasmids with LacZa insert for blue-white screening. E.coli friendly SEVA plasmids were used as backbones and LacZa insert was taken from the original GB 2.0 pDGB1 vectors [2]. Resulting in vectors with the pDGB1 cassete and restriction enzymes (BsaI, BsmBI). pDGB1 vectors have features for plants. On the other hand SEVA plasmids are applicable in all procaryotes [2]. SEVA plasmids are segmented in parts with rare restriction enzymes such us AscI, FseI, PashAI, PacI bordering the features of the plasmid. Two restriction enzymes are between the antibiotic, oriT, replication,etc. Each plasmid can be deconstructed and reconstructed with desirable parts. Also SEVA plasmids are readable in SBOL (Synthetic Biology Open Language).

Experimental Use and Experience

The following parts were cloned in this vector BBa_K3505025 BBa_K3505035

pDGB1 a1R verification with no insert

Fig.3: pDGB1 a1R with no insert PCR with sequncing primers. Expected band 750bp (LacZa)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3118
    Illegal XbaI site found at 3091
    Illegal PstI site found at 3079
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3118
    Illegal PstI site found at 3079
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3118
    Illegal BamHI site found at 11
    Illegal BamHI site found at 2858
    Illegal BamHI site found at 3097
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3118
    Illegal XbaI site found at 3091
    Illegal PstI site found at 3079
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3118
    Illegal XbaI site found at 3091
    Illegal PstI site found at 3079
    Illegal NgoMIV site found at 1290
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3461
    Illegal BsaI.rc site found at 2879

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
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