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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?

• 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.

• 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.

• The ability of cutting and pasting several parts by using a single restriction enzyme and a ligase makes the whole assembly easier to perform.

• No scars are left when assembling the different parts. If the overhangs are carefully designed, scarless DNA junctions can be obtained.

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:

This is the GG reaction needed for the adaptation of any DNA sequence to the GG standard. It implies the removal of internal restriction sites for the enzymes used in GG (BsaI, BsmBI) and the addition of appropriate 4-nt flanking overhangs to convert a single level 0 part (promoter, RBS, CDS or terminator) into a standard part inside a predesigned vector (domestication to the GB grammar).

We are using this level 0 assembly in the lab, so that we domesticate every single part which Printeria will use to create its own transcriptional units. The goal is to end up with a series of plasmids that contain each of the different promoters, RBSs, CDSs and terminators.

In our specific case, sticky ends of the parts are predesigned so that upon cleavage with BsmBI, they are pasted into our domestication vector BBa_P10500 in a proper way.

The BBa_P10500 vector has a chloramphenicol resistance and the lacZ cassette so that blue-white screening can be performed among the transformed E. coli cells.

Figure 1: Designing of the different basic parts. BsmBI restriction sites are represented by the yellow and black puzzle-like pieces. 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 be correctly positioned.

Figure 2: BBa_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. Yellow and black puzzle-like pieces represent the cleavage sites for BsmBI..

Figure 4: Domestication of a promoter inside the BBa_P10500

Figure 5: Basic domesticated part. Light yellow and grey blocks represent the BsmBI sticky ends which have been ligated. As the new plasmid is assembled, BsaI restriction sites appear (blue and pink puzzle-like pieces).

Figure 6: Golden Braid compatible level 0 parts. BsaI restriction sites appear (coloured puzzle-like pieces).

Level 1 Assembly

This second level of complexity cannot be performed without having fulfilled the domestication of the parts (LEVEL 0). Once it is done, we can create a simple transcriptional unit.

As said before, each domesticated parthas a BsaI recognition site and a cleavage site which, when cleaved, will match with the contiguous parts. In other words, promoters will stick with the left end of our destination vector, pGreen alpha1 (KanR), using their left sticky ends, and with the left end of the RBSs using their right ends. At the same time, CDSs will stick to the right end of these RBSs using their left sticky ends, and to the left end of the terminators with their right ends. Finally, the terminators will stick to the right end of our backbone destination vector with their right ends, so that we will end up having a plasmid with a single TU inside it.

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: pGreen alpha 1 destination vector. The puzzle-like pieces represent the restriction sites for BsaI. It has kanamycin resistance.

Figure 8: BsaI digested destination and domesticated part to build a transcriptional unit.

Figure 9: Transcriptional unit assembly

Figure 10: TU insertion inside pGreen alpha1

Figure 11: Light coloured sequences represent the BsaI sticky ends which have been glued. As the new plasmid is assembled, BsmbI restriction sites appear (blue and dark blue).

Level 2 Assembly

This is the last level of complexity in which, by using the combination of the α and Ω vectors, we can cut and paste several transcriptional units inside the same plasmid so that more complex genetic circuits can be created.

Printeria aims to arrive to this level of complexity someday making its possibilities and combinations infinite. This will be Printeria’s FUTURE.

References

1. Shetty RP, Endy D, Knight TF. Engineering BioBrick vectors from BioBrick parts. J Biol Eng. 2008;2: 5.

2. Andreou AI, Nakayama N (2018) Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly. PLOS ONE 13(1): e0189892.

3. 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.

4. 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