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. 2018 Jan 2;13(1):e0189892. doi: 10.1371/journal.pone.0189892

Fig 2. Mobius Assembly framework.

Fig 2

(A) Mobius Assembly uses a two-level (Level 1 and 2) approach for transcriptional unit (TU) and multi-TU augmentation. Each level is comprised of four Acceptor Vectors. The four Level 1 Acceptor Vectors (A, B, Γ, and Δ) carry spisPink gene as the visible cloning screening marker and confer Kanamycin resistance. The four Level 2 Acceptor Vectors (A, B, Γ, and Δ) carry sfGFP gene as the visible cloning screening marker and confer Chloramphenicol resistance. The standard parts stored in mUAVs are released and fused in a Level 1 reaction to form a TU. Up to four Level 1 TUs can be fused in a Level 2 reaction to form a multi-TU cassette. Switching back and forth between Level 1 and 2 leads to further expansion of multi-TUs according to the geometric sequence: 1, 4, 16, 64,…. Red arrows denote AarI restriction sites and Purple arrows BsaI restriction sites. (B, C, and D) E. coli colonies carrying mUAV (B), Level Acceptor 1 Vector A (C), and Level 2 Acceptor Vector A (D), which respectively exhibit purple, magenta and yellow colour after overnight incubation. Successful assembly produces white colonies.