Part:BBa_K3505043

pUPD2 BACKBONE for Golden Braid Cloning

This backbone combined with the insert BBa_K3505041 results in this comlpete Vector BBa_K3505007

• The Backbone has Chloaramphenicol Resistance as the Level 0 GoldenBraid parts have. Ori pMB1 for high replication of DNA.
• The insert has LacZa module for blue white screening. Proper BsmBI and BtgZI restriction sites are flanking the LacZa module for the modular cloning.

Level 0 vector for GoldenBraid assembly. The Level 0 modules are Interchangable parts in order everyone can use it as it is without any modification based on GoldenBraid grammar.

Figure 1. The GB2.0 grammar

Figure 2. pUPD2 map. Features and Restriction Sites.

Figure 3. pUPD2

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 build 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 is derived from iGEM-borne pSB1C3) for any GBpart 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 ‘Level alpha’ (Level a)

But this is not the end, GoldenBraid outperforms GoldenGate (which everyone knows) because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression GoldenGate. Bianry 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.

Experimental Use and Experience

The following parts were cloned in this vector BBa_K3505003 BBa_K3505005 BBa_K3505006 BBa_K3505012 BBa_K3505013 BBa_K3505014 BBa_K3505015 BBa_K3505016 BBa_K3505017 BBa_K3505018 BBa_K3505019 BBa_K3505020 BBa_K3505021 BBa_K3505022 BBa_K3505023 BBa_K3505024

pUPD2 verification with no insert

Fig.4: pUPD2 with no insert digested with EcoRI. Expected bands 2414bp, 276bp

Source

Our advisor Nikolaos Ntelkis provided us the pUPD2 vector.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 2061
  Illegal SpeI site found at 14
  Illegal PstI site found at 28
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2061
  Illegal SpeI site found at 14
  Illegal PstI site found at 28
  Illegal NotI site found at 21
  Illegal NotI site found at 2067
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2061
  Illegal XhoI site found at 1045
  Illegal XhoI site found at 1937
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2061
  Illegal suffix found in sequence at 14
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2061
  Illegal XbaI site found at 2076
  Illegal SpeI site found at 14
  Illegal PstI site found at 28
  
• 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