Plasmid

Part:BBa_K3505010

Designed by: Venetios Michelioudakis   Group: iGEM20_Thessaly   (2020-10-20)


pDGB1 omega 1R (Ω1R) for Bacterial GoldenBraid

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

Design Notes

This vector was designed to be Ω1. After sequencing we saw that the LacZa cargo was inserted in the opposite orientation so this is an Ω1R. This DOSEN'T AFFECT the cloning. We reasure that because we cloned construdts in this vector.

In order to result in an alpha vector an omega 1 and an omega 2 must be combined. So our TUs are some in the Ω1R some in Ω2 and some in both.

Fig.1: Ω1R plasmid Features and Restiction Sites.

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 pUPD2 BBa_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).

Design Notes

In order to result in an alpha vector an omega 1 and an omega 2 must be combined. So our TUs are some in the Ω1R some in Ω2 and some in both.

Experimental Use and Experience

The following parts were cloned in Ω1R BBa_K3505036 BBa_K3505037

pDGB1 Ω1R verification with no insert

Fig.2: pDGB1 omega1R Digested with BamHI. Expected bands 3153, 382, 239

Source

Christos Batianis [3]

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3097
    Illegal EcoRI site found at 3387
    Illegal XbaI site found at 3360
    Illegal PstI site found at 6
    Illegal PstI site found at 3348
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3097
    Illegal EcoRI site found at 3387
    Illegal PstI site found at 6
    Illegal PstI site found at 3348
    Illegal NotI site found at 3103
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3097
    Illegal EcoRI site found at 3387
    Illegal BamHI site found at 3127
    Illegal BamHI site found at 3366
    Illegal BamHI site found at 3748
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3097
    Illegal EcoRI site found at 3387
    Illegal XbaI site found at 3360
    Illegal PstI site found at 6
    Illegal PstI site found at 3348
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3097
    Illegal EcoRI site found at 3387
    Illegal XbaI site found at 3360
    Illegal PstI site found at 6
    Illegal PstI site found at 3348
    Illegal NgoMIV site found at 1019
    Illegal NgoMIV site found at 1421
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3133
    Illegal BsaI.rc site found at 3742

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
  • [3]Damalas, 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

Biotechnology, 13(6), pp.1793-1806.

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