Team:Valencia UPV/Design
Biological Design: Golden Gate assembly
Introduction
We are continuously talking about Printeria as a machine which can create its own genetic circuits, by using pre-designed parts, and ‘print’ them inside different living cell chassis. But how is Printeria going to perform all these complex reactions?
One of the first attempts to standardize a restriction enzyme-based DNA assembly method was BioBricks (1). However, its pairwise nature can make the construction of multipart systems, such as transcriptional units, time-consuming. To solve this, Printeria is using a state-of-the-art technology based on the Golden Gate Assembly, the GoldenBraid Assembly Method. This technology uses type IIS restriction enzymes in order to cut all the parts and build these genetic circuits. What makes Golden Gate the most suitable DNA assembly method for Printeria?
Golden Gate uses Type IIS restriction enzymes, are a group of endonucleases that recognize specific asymmetric double stranded DNA sequences and cleave outside of their recognition sequence. Thus, digestion leaves short single stranded overhangs with non-specific sequences. This allows us to define the cleavage sequence of each part enabling the assembly of multiple fragments of DNA in a single reaction. This is the way in which directionality is maintained and parts are assembled in the desired order.
But why is this assembly technique so crucial for our machine to work?
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The design of the entry and destination vectors with type IIS recognition sites in opposite directions leads into a final plasmid - once the DNA construction has been ligated - where there is no recognition site. So, once the insert has been ligated, it cannot be cut again. This allows simultaneous digestion and ligation in a one-pot reaction so that the whole assembly is taking place in a single step. This fact makes the Golden Gate Technology perfect for our machine to work, as the whole reaction should take place in a single droplet.
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Robust reaction. Small modifications on the temperatures, number of cycles or reaction time of the Golden Gate protocol result in less efficient but still successful assemblies. Therefore, the moving of the droplet across the PCB surface as well as slight variations in the temperature during the reaction should not be a real problem for it to work.
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The ability of cutting and pasting several parts by using a single restriction enzyme and a ligase makes the whole assembly easier to perform.
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No scars are left when assembling the different parts. If the overhangs are carefully designed, scarless DNA junctions can be obtained.
The Golden Braid Assembly method
GoldenBraid relies on Golden Gate for the assembly of transcriptional units and introduces a double-loop strategy for the assembly of multigenic constructs. In the GG assembly method the transcriptional units can be combined in binary steps to create multigene structures (several TUs within the same destination plasmid). To do so, this system relies on the switching between two levels of plasmids, α and Ω , with different antibiotic resistance.
The assembly process with GG can be divided into three different steps:
Level 0 Assembly
This is the GB reaction needed for the adaptation of any DNA sequence to the GB standard. It implies the removal of internal restriction sites for the enzymes used in GB (BsaI, BsmBI) and the addition of appropriate 4-nt flanking overhangs to convert a single basic part (promoter, RBS, CDS or terminator) into a standard part (domestication to the GB grammar, Figure 1). These pieces are then subcloned inside the BBa_P10500 vector (Figure 2).
We have carried out this Level 0 assembly in the lab, as we had to domesticate every single part of our Printeria DNA Basic Part Collection.
The goal is to end up with a series of plasmids that contain each of the different promoters, RBSs, CDSs and terminators.
To do so, sticky ends of each basic part are predesigned so that upon cleveage with BsmBI (Figure 3), they are pasted into our domestication vector pUD2 in a proper way (such as in Figure 4 and 5 for a promoter domestication). As a result, standard overhangs, characteristics for each type of basic piece, are ready for the next level of assembly (Figure 6).
In this assembly, BBa_P10500 plasmid has a chloramphenicol resistance gene and the lacZ cassette, so that blue-white screening is performed to select the recombinant colonies among the transformed E. coli cells.
Figure 1: Designing of the different basic parts. The upper sequence corresponds with the strand that was ordered for synthesis. The lower sequence represents the complementary strand. BsmbI restriction sites are represented by the yellow and black cuts. The coloured sequences represent BsaI restriction sites when the part is inserted in our domestication vector. A 6-nucleotide scar was added to the RBS so that the ribosome could bind.
Figure 2: P10500 domestication vector. Yellow and black puzzle-like pieces represent the restriction sites for BsmbI. It has chloramphenicol resistance.
Figure 3: BsmbI digested part and vector.
Figure 4: Domestication of a promoter inside the P10500
Figure 5: Basic domesticated part. Light yellow and grey sequences represent the BsmbI sticky ends which have been glued. As the new plasmid is assembled, BsaI restriction sites appear (blue and pink cuts).
Figure 6: All Golden Braid compatible domesticated parts. BsaI restriction sites appear. They are represented by the coloured puzzle-like pieces.
Level 1 Assembly
This second level of complexity cannot be performed without having fulfilled the domestication of the parts. Once it is done, we can now create a simple transcriptional unit. Thus, this is the level of complexity that Printeria does.
As explained before, each of the domesticated parts now has a BsaI recognition site and so a standardized cleaving sites which, when cut, will be complementary with the contiguous part. This construction is assembled into an alpha 1 GB destination vector with the kanamicine resistant gene (Figure 7). When cleaving both Golden Braid adaptated basic parts and this alpha1 vector, complementary overhangs allows the directional assembly and subsequent insertion of the TU (Figures 8, 9 and 10).
After the Golden Braid one-step reaction, the recombinant plasmid has BsmBI endonuclease recognition sites flanking the insert sequence, so this construction could be then cleveaged to design multigenetic constructions with the GB Level 2 (Figure 11).
Figure 7: GoldenBraid alpha 1 destination vector. The rational positioning of the BsmbI recognition sites allows to carry out the Level 2 assembly.
Figure 8: BsaI digestion of both Level 1 destination vector and a GoldenBraid basic part.
Figure 9: Transcriptional unit assembly
Figure 10: TU insertion into the alpha 1 destination vector
Figure 11: Light coloured sequences represent the Golden Braid scars as a consequence of the complementary sticky ends joined. BsmbI recoginition sites (blue and dark blue sequences) allow to subclone the TU through the Level 2 GB assembly.
Level 2 Assembly
This is the last level of complexity in which, by using the combination of the α and Ω vectors, it is posssible to assemble several transcriptional units into the same plasmid, so creating multipartite genetic constructions.
To do so, two independent transcriptional units must be assembled into an alpha1 and alpha 2 vector, respectively, so they can be then introduced into an omega destination vector with a BsmBI one-pot reaction. Similarly, it is possible to join two omega genetic contructions into an alpha level vector. Thus, by switching between this two levels of plasmids more and more complex multigenetic constructions can be progressively ligated in a hierarchical way.
Printeria's future aim is to arrive to this level of complexity, so that complex genetic circuits could be 'printed' in an affordable and automatical way.
References
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Shetty RP, Endy D, Knight TF. Engineering BioBrick vectors from BioBrick parts. J Biol Eng. 2008;2: 5.
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Andreou AI, Nakayama N (2018) Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly. PLOS ONE 13(1): e0189892.
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Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, et al. (2011) GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLOS ONE 6(7): e21622.
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Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, et al. Golden- Braid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. Plant Physiol. 2013; 162: 1618–1631