Team:BostonU/MoCloChara

As mentioned above, our MoClo system has three levels of assembly.

Level 0: Basic DNA Parts (ex: promoter, gene, etc.) are PCR amplified and cloned into MoClo destination vectors to form Level 0 Parts. During the PCR reaction, the correct fusion sites and BpiI sites are added to either side of the DNA sequence. The orientation of the BpiI sites is crucial, where the 3’ BpiI site is inverted while the 5’ BpiI site is not (Fig. 3). This PCR reaction is mixed with its matching Destination Vector in a MoClo reaction. After a transformed clone has been confirmed, this plasmid is now called a Level 0 Part and has the following order of DNA sequences: BsaI site-Fusion site 1-DNA Part-Fusion site 2-BsaI inverted (Fig. 3). Notice that BpiI is removed from the final product, thus requiring the Destination Vector to contain both BpiI and BsaI flanking the fusion sites (Fig. 3).







Figure 3: Construction of a Level 0 MoClo Part. PCR primers are designed as shown in the top of the figure to include 2bp (NN), then the 6bp BpiI site (in purple), 2bp (NN), then the 4-bp fusion site (in blue), followed by 18-24bp of the DNA part of interest. Once amplified with high fidelity DNA polymerase, the PCR product is mixed with the Level 0 Destination Vector that has matching fusion sites in a MoClo reaction to create the final Level 0 MoClo Part shown in the final plasmid.



Level 1: Up to six Level 0 Parts are ligated together to form Level 1 Parts. In our lab, Level 1 Parts most often result in complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Parts are flanked by BpiI sites and two different 4pb-fusion sites (Fig. 4).





Figure 4: Construction of a Level 1 MoClo Part. Four basic DNA Parts (promoter, RBS, CDS, and terminator) are each shown in green vectors to denote the chloramphenicol resistance cassette and each has two 4bp non-palindromic fusion sites flanking the DNA Part. The fifth vector is the Destination Vector for the final construct, which is orange to denote the kanamycin resistance cassette and contains lacZ for blue-white screening. The BsaI sites (red text) are shown in the DNA Part vectors and Destination Vector with the direction they cut (red arrow). The 4bp fusion sites (blue text) shown in the five top plasmids indicate the fusion sites that will remain with either the DNA Parts or the Destination Vector after the BsaI digestion.


Level 2: Up to six Level 1 Parts are ligated together to form Level 2 Parts. More complex circuits, such as an inverter or NOR gate, can be built using Level 2 Parts. Like Level 0 Parts, Level 2 Parts are flanked by BsaI sites and two different 4pb-fusion sites.

References

[1] Weber et al. (2011) "A Modular Cloning System for Standardized Assembly of Multigene Constructs." PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765
[2] Engler et al. (2009) "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes." PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553
[3] Haddock, T., and Densmore, D. (2013) BBF RFC 94: The Modular Cloning Assembly: Standardized Assembly of Bacterial Transcriptional Units Using Type IIS Restriction Enzymes BsaI and BpiI.