To explore the fundamentals of modular cloning using the popular Golden Braid cloning standard via the completely free ApE Plasmid Editor. Students will be able to domesticate genetic parts into the Golden Braid 2.0 syntax, assemble Transcription Units (TU’s) made of several standardized parts in a single tube reaction, and chain multiple TU’s together into biosynthetic gene clusters or modules (MODs).
Molecular cloning has been around for quite some time now, and has enabled researchers to stitch together custom bits of DNA into functional circuits to augment the genotype and/or phenotype of the recipient of said DNA. Traditionally, molecular cloning is done using E. coli plasmids and restriction endonuclease enzymes (RE’s) which recognize short DNA sequences on the plasmid and cut within the boundaries of said sequence, opening the closed loop of plasmid DNA at one or more locations where the ends of each resulting fragment are chemically reactive in the presence of DNA Ligase enzymes. A textbook example is that of the venerable EcoR1 enzyme:
https://media.sciencephoto.com/image/f0374514/800wm/F0374514-EcoRI_enzyme_restriction_site,_illustration.jpg
The recognition sequence for the EcoRI enzyme is 5’-GAATTC-’3 and it cuts in a zig-zag pattern forming phosphorylated “sticky ends”, that are then capable of being ligated together using DNA Ligase enzymes which fuse compatible ends. Genetic sequences cut by the same Restriction Endonuclease can be ligated together. A traditional cloning strategy is to flank your sequence of interest with unique restriction enzyme sites that appear only once on your plasmid. Through this process you can orient sequences in a desired fashion that maintains genetic grammar rules, with some exceptions we will elaborate on soon. As an example, I use a traditional cloning scheme that is as follows:
EcoR1-Promoter-NcoI-CodingSequence-SacI-Terminator-KpnI
Where EcoRI, NcoI, SacI, and KpnI are restriction sites that each only appear once on my plasmid. This cloning strategy allows for the assemble of a single Transcription Unit (TU), but if I wish to expand this TU to add more coding sequences as in an operon, multiple TU’s chained together to make modules (MOD’s), or generally modify the plasmid, I would need to find two new unique cutsites for each additional genetic part. You could save on cut sites by using the same unique enzyme cut site on either end of your new genetic part like this:
EcoRI-Promoter-EcoRI-NcoI-CodingSequence-NcoI-SacI-Terminator-SacI
With this strategy you save on available cut sites, but you cannot ensure the orientation of each part during cloning since the part is now clonable in either direction due to having the same cut site on both ends. This will cause issues where your part inserts into the plasmid in the reverse orientation like so:
EcoRI-Promoter-EcoRI
or EcoRI-retomorP-EcoRI
Another drawback of Traditional Cloning is that you often limited in how many parts you can clone at one time, dictated by the number of unique cut sites and available enzymes, which makes multi-part assemblies very expensive and/or take weeks of cloning cycles to complete. In a typical cloning cycle, you must:
