pDGB1 omega 2 (Ω2) for Bacterial GoldenBraid

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

Fig.1: Ω2 plasmid Features and Restiction Sites.

Usage and Biology

GoldenBraid (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, GoldenBraid 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.

pDGB1 Ω1R verification with no insert

Fig.2: U3 C3 pDGB1 omega2 Digested with EcoRV and HidIII. Expected bands 2900, 398, 223

Source

Christos Batianis [3]

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 3277
Illegal XbaI site found at 3304
Illegal PstI site found at 3316
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 3277
Illegal PstI site found at 3316
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 3277
Illegal BamHI site found at 3298
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 3277
Illegal XbaI site found at 3304
Illegal PstI site found at 3316
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 3277
Illegal XbaI site found at 3304
Illegal PstI site found at 3316
Illegal NgoMIV site found at 948
Illegal NgoMIV site found at 1350
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2918
Illegal BsaI.rc site found at 6

References