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The modular Golden Gate cloning technique SapTrap to assemble C. elegans MosSCI transgene vectors (A) Gene fragments are cloned into the pCR-Blunt II-TOPO Vector to create donor cassette plasmids. Note that gene fragments are flanked by a SapI recognition site (red) and homology sequences (blue) that define the position in the final MosSCI transgene vector. (B) Modular assembly of the MosSCI transgene vector. Individual gene fragments are liberated from the donor cassettes by SapI digestion, while the 5 0 -homology sequences define the position of the gene fragments in the final MosSCI transgene vector. Note that the SapI recognition sites are oriented such that upon digestion they are removed from the individual gene fragments and vector backbone. (C) Overview of primer design for SapTrap, for the 4 cassette or 5 cassette system.
Source publication
The relative positioning of organelles underlies fundamental cellular processes, including signaling, polarization, and cellular growth. Here, we describe the usage of a light-dependent heterodimerization system, LOVpep-ePDZ, to alter organelle positioning locally and reversibly in order to study the functional consequences of organelle positioning...
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... the above in mind, we recommend the recently developed Golden Gate cloning technique SapTrap to assemble C. elegans MosSCI transgene vectors (Fan et al., 2019) carrying LOVpep and ePDZ constructs in a fast, efficient, inexpensive, and scar-free manner ( Figure 2). The MosSCI backbone and donor plasmids carrying LOVpep and ePDZ that are compatible with this method are available at Addgene ( Fan et al., 2019). ...
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... Find templates encoding the parts you want to combine and order primers to produce gene fragments flanked by SapI restriction sites. Key in the SapTrap method is that SapI cuts DNA at defined positions adjacent to its recognition sequence to generate three-base 5 0 overhangs ( Figure 2, red sequence/bars=recognition site, blue sequence/bars= three-base 5 0 overhangs). By designing SapI restriction fragments with complementary overhangs, multiple fragments can ll ...
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... assembled together in a defined order in a single digestion and ligation reaction ( Figure 2B, the defined order is illustrated by the fragments ranging from light gray to dark gray). Primer design and examples of primers can be found in Figure 2C and in the study by Fan et al. (2019). ...
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... assembled together in a defined order in a single digestion and ligation reaction ( Figure 2B, the defined order is illustrated by the fragments ranging from light gray to dark gray). Primer design and examples of primers can be found in Figure 2C and in the study by Fan et al. (2019). If templates are not available, codon-optimized sequences can be ordered. ...
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... you amplify your gene fragments with your primers and template DNA, use a high-fidelity PCR kit as per the manufacturer's instructions. 9. Generate donor plasmids by cloning the PCR products or gBlocks into the pCR BluntII vector backbone using the Zero Blunt Topo system (Thermo Fisher Scientific), as per the manufacturer's instructions ( Figure 2A Pause point: Bacterial colonies can be stored at 4 C for up to 2-3 weeks. ...
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... Protocols 2, 100273, March 19, 202113. MosSCI targeting vectors are assembled using the SapTrap method in a single tube ( Figure 2B) using the following protocol: add 1 mL 50 nM pXF87 (MosSCI backbone, available at Addgene), 1 mL 50 nM of each donor cassette plasmid, 5 mL 103 NEB cutsmart buffer, 5 mL 10 mM ATP (not dATP), 1 mL SapI enzyme (10 units), 1 mL T4 DNA ligase (400 units) and ddH 2 O to a final volume of 50 mL. Incubate this reaction mixture 5 min at 37 C (=SapI digestion) and 5 min at 16 C (ligation), repeating this for a total of 35 cycles. ...
