Biomedical Engineering Reference
In-Depth Information
level
plasmids are designed to have their sites in inverted
orientations. Plasmids also carry different resistance markers for
effi cient counter selection.
Although in the most basic setup only four pDGBs are needed
to establish the double loop, we build eight different pDGBs in
order to make possible the assembly of TUs in reverse orientation.
Numbers and letters serve to identify each destination plasmid
according to the fl anking overhangs left by BsaI and BsmBI diges-
tion, respectively:
Ω
1. Level
plasmids are used as destination plasmids in BsaI
GB-reactions. These are pDGB_A12C, pDGB_C12B, pDGB_
A21C, and pDGB_C21B. In most cases only pDGB_A12C or
pDGB_C12B will be used unless there is any interest in assem-
bling TUs in reverse orientation (in this case the choice would
be pDGB_A21C or pDGB_C21B). This group of plasmids
contains kanamycin as resistance marker and is both used
for the multipartite assembly of GBparts and for the binary
combination of TUs.
2. Level
α
plasmids are used as destination plasmids in BsmBI-GB
reactions. These are pDGB_1AB3, pDGB_3AB2, pDGB
1BA3, and pDGB 3BA2. Regularly, only pDGB_1AB3 or
pDGB_3AB2 will be selected by users unless they need to
assemble TUs in reverse orientation. This group of plasmids
incorporates spectinomycin as bacterial resistance.
Ω
The mechanism of GB is shown in Fig.
1
. Standard parts are
normally assembled in level
plasmids, for example into pDGB_
A12C. The resulting composite parts can be combined with other
structures assembled in the complementary pDGB_C12B using
any of the level
α
plasmids as destination vectors for the assembly.
In a second assembly round, composite parts assembled using level
Ω
Ω
plasmids
provided that they share a common sticky end. As it can be
observed, GB works as an endless iteration of binary assemblies
keeping the ability of incorporating more units, with the only the-
oretical limit of the capacity of the destination vector backbone.
plasmids can be combined together inside a level
α
“GBparts” are functional DNA fragments fl anked by fi xed 4 nt
overhangs which are generated by cleavage with the type IIS
enzyme BsaI. As described in Golden Gate assembly (Chapter
9
)
[
10
], the overhangs determine the relative position of a GBpart in
a multipartite assembly. In the most general case, we considered
three functional standard categories when building TUs: promot-
ers (which include the transcription origin and 5
2.2 GoldenBraid
Parts
′
UTR), coding
regions (CDS), and terminators (which include 3
UTR and polyA
site and transcriptional stop signal). Each category corresponds
to a relative position in the assembly (although users can defi ne
′
