Figure 1.
General overview of the hierarchical and modular cloning system.
(A) Libraries of basic (level 0) modules contain cloned and sequenced genetic elements such as promoters (P), 5′ untranslated regions (U), signal peptides (SP), coding sequences (CDS) and terminators (T). Transcription units are assembled from selected level 0 modules using a one-pot one-step cloning reaction. Multigene constructs are then assembled in a second cloning step (and optionally further steps) from the transcription units. (B) Level 0 modules of different classes are flanked by compatible fusion sites. Each fusion site consists of 4 nucleotides of choice (boxed) flanked by a type IIS enzyme recognition site on the left or right side (vertical box drawn under the fusion site). (C) Examples of assembled transcription units for secreted or cytosolic proteins. The transcription unit for the cytolic protein was assembled from 4 modules rather than 5, using a CDS module cloned between fusion sites AATG and GCTT.
Figure 2.
Level 0 destination vectors and principle for removal of internal sites from level 0 modules.
(A) Level 0 destination vectors. Level 0 modules are made by amplification of selected sequences with primers adding flanking BpiI sites, and cloning of the amplified fragment (shown above the horizontal dotted line) via BpiI into the designated level 0 destination vectors (shown below). In addition to the 5 basic destination vectors, pL0-P, pL0-U, pL0-S, pL0-C and pL0-T, additional destination vectors allow cloning several genetic elements in one module. For example, plasmid pL0-SC can be used to clone sequences encoding cytosolic proteins, which do not contain a signal peptide. (B) Strategy for removing internal type IIS recognition sequences. Removal of a BsaI site in a fragment of interest is done by amplifying two fragments with primers pr1 and 2 and primers pr3 and 4. Primers pr2 and pr3 span the BsaI recognition site and introduce a single nucleotide mismatch (indicated by an arrow). As all primers have BpiI recognition sites in their 5′ extensions, the PCR fragments are cloned with a BpiI-based Golden Gate cloning reaction in the appropriate level 0 destination vector.
Figure 3.
Arrangement of type IIS restriction sites and fusion sites for all assembly levels.
A detailed overview of the organization and orientation of the type IIS restriction sites and the fusion sites at the different levels of the MoClo system is shown. A PCR product containing a promoter flanked by BpiI recognition sites and promoter-specific fusion sites (highlighted with color) is cloned via BpiI into the level 0 destination vector pL0-P. The promoter fragment in the resulting level 0 module is still flanked by the same fusion sites, but can now be released with BsaI. The level 0 promoter module and the other level 0 modules required to form a complete transcription unit (not shown) are then assembled via BsaI into a level 1 destination vector. As the fusion sites created by BsaI and BpiI do not overlap, the assembled level 1 module (here TU1) is equipped with two level 1-specific fusion sites (TGCC and GCAA). The level 1 module TU1 and other level 1 modules of choice (TU2 and not shown) can then be assembled via BpiI into the final level 2 construct in which no type IIS recognition sites are left. n indicates that any nucleotide can be used. CRed, red color selectable marker; P, promoter module; TU, assembled transcription unit.
Figure 4.
(A) Constructs are assembled by mixing in one tube all module plasmids (or PCR fragments for level 0) and a destination vector together with the appropriate type IIS enzyme (indicated above the arrows) and ligase. ++ indicates that only one of several modules was drawn due to space limitation. Each fusion site is shown as a box indicating its 4 nucleotides; the two boxes below show which type IIS recognition sites flank the fusion sites on the left and/or right sides. P1-a/b, promoter fragment a or b; U, 5′ untranslated region; SP, signal peptide; CDS, coding sequence; T, terminator; CRed, red color selectable marker; LacZ, lacZα fragment, blue color selectable marker; L2E, end-linker 2; ApR, ampicillin resistance; KmR, kanamycin resistance; SpR, spectinomycin resistance. (B) General structure of level 2 constructs. Transcription units are located between the sequences GGAG and CGCT (remnants of fusion sites used for assembly of transcription units in forward orientation) or AGCG and CTCC (for transcription units cloned in reverse orientation). The number above the transcription units indicates the relative position of the transcription units in the final construct (indicates which of the 7 level 1 destination vectors shown in figure 5 was used for assembly of this transcription unit). The construct is terminated at the right end by an end-linker (EL) that joins the downstream fusion site of the last transcription unit (NNNN) with the downstream fusion site of the destination vector (GGGA).
Figure 5.
Vector set required for the MoClo system.
All level 1 destination vectors (forward and reverse), level 2 destination vectors and the different end-linker sets are shown. Dotted arrows indicate the linear relationships between fusion sites in level 1 destination vectors. Compatible fusion sites are labeled with the same color.
Figure 6.
The MoClo cloning principle can be repeated indefinitely.
Every cloning step relies on three elements that are different from one level to the next: antibiotic selectable marker, type IIS enzyme(s), and color selectable marker. Cloning after level 2i-1 requires the simultaneous use of two type IIS enzymes: BpiI/BsaI or BpiI/Esp3I.
Figure 7.
Structure of the eleven level 1 modules (A) and the final level 2 construct cL2-13 (B).
All transcription units were assembled from 5 plasmids: 4 level 0 modules (promoter, 5′ untranslated region, CDS, and terminator) and a destination vector. All proteins are cytosolic except the two from constructs cL1-8 and 9 which are secreted. For both of these, the coding sequence module already contained the signal peptide. LB, T-DNA left border; RB, T-DNA right border; Ω, tobacco mosaic virus 5′ untranslated region; genetic elements used are listed in Table S1.
Figure 8.
Cloning efficiency of level 1 and 2 constructs.
(A) Assembled transcription units are schematically represented as boxes annotated with the name of the CDS they contain. Transcription units shown in grey were cloned in the previous step (in construct cL2-6). The respective cloning position of each transcription unit is indicated on the top. For level 2 constructs, the end-linker is shown as a circle. The number of colonies obtained per transformation is shown by color type, with the first number corresponding to the expected correct constructs (for cL2-6, wrong clones could be either red or green). (B) Plates from transformation of constructs for level 2-1 (cL2-2, cL2-4 and cL2-5) and level 2i-1 (cL2-6). Since level 2-1 cloning uses red/white selection, the correct constructs should be white, while colonies containing the original destination vector construct should be red. Level 2i-1 uses a blue/red selection, with colonies containing correct constructs expected to be blue, whereas incorrect ones can be red or green (contain both the canthaxanthin operon and the lacZα fragment).
Figure 9.
Expression of GFP by level 2 constructs.
Level 2 constructs in Agrobacterium tumefaciens were inoculated into N. benthamiana leaves. GFP expression was observed at 5 dpi under UV light. The number in parenthesis indicates the number of transcription units in each infiltrated construct.