Part:BBa_K3505007

pUPD2 for Golden Braid

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.LacZa module as cargo for Blue White Screening between 2 BsmBI sites and 2 BtgzI sites. Resistance in Chloramphenicol and ori pMB1 for high replication of the DNA.

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 Golden Gate. 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 EcoRI site found at 260
  Illegal EcoRI site found at 2674
  Illegal XbaI site found at 233
  Illegal SpeI site found at 627
  Illegal PstI site found at 221
  Illegal PstI site found at 641
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 260
  Illegal EcoRI site found at 2674
  Illegal SpeI site found at 627
  Illegal PstI site found at 221
  Illegal PstI site found at 641
  Illegal NotI site found at 634
  Illegal NotI site found at 2680
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 260
  Illegal EcoRI site found at 2674
  Illegal BamHI site found at 239
  Illegal XhoI site found at 1658
  Illegal XhoI site found at 2550
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal suffix found in sequence at 627
  Illegal EcoRI site found at 260
  Illegal EcoRI site found at 2674
  Illegal XbaI site found at 233
  Illegal PstI site found at 221
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 260
  Illegal EcoRI site found at 2674
  Illegal XbaI site found at 233
  Illegal SpeI site found at 627
  Illegal PstI site found at 221
  Illegal PstI site found at 641
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 14
  Illegal BsaI.rc site found at 611
  

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