Difference between revisions of "Team:Valencia UPV/Design"

Biological Design: Golden Braid assembly

Introduction

We are continuously talking about a device 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. However, its pairwise nature can make the construction of multipart systems, such as transcriptional units, really time-consuming. As a consequence, we needed a multipartite assembly method capable of joining several DNA basic parts in a fast but also simple way.

Thus, Printeria is using a state-of-the-art technology based on the Golden Gate Technology: the Golden Braid assembly method. This technology uses type IIS restriction enzymes in order to cut DNA basic parts and so build genetic circuits constructions.

The Golden Gate assembly is based on type IIS enzymes. But what does this really mean?

Type IIs restriction enzymes are a group of endonucleases that recognize asymmetric double stranded DNA sequences and cleave outside its recognition sequences. Thus, digestion leaves short single stranded overhangs with non-specific sequences.

This allows us to design the cleaving region so that the sticky ends of the parts to join are complementary to each other. By this way, directionality is maintained and parts are assembled in the desired order.

But why is this assembly technique so crucial for Printeria?

• Carefully positioning the recognition and cleavage sites, in opposite directions, for the entry and destination vectors 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. This fact makes the Golden Braid method perfect for Printeria to work, as the whole reaction can take place in a single droplet.

• Robust reaction. As a consequence, the moving of the droplet across the PCB surface should not be a real problem to carry out the assembly reaction effectively.

• The ability of cutting and pasting several parts by using the same enzymes makes the whole assembly step easier to perform.

• Thanks to the user design of the DNA basic parts overhangs, it is an almost scarless assembly process

In the GB assembly method the transcriptional units can be combined in binary steps to grow 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.

This Technology is then divided into three different complexity levels:

This is the easiest and first step Golden Braid reaction. 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 standardiced GB 3.0 piece, which is subcloned inside the BBa_P10500 vector (domestication to the GoldenBraid grammar).

We have carried out this Level 0 assembly in the lab, as we have domesticated every single part of the Printeria DNA basic part collection

The goal is to end up with the different promoters, RBS, CDS and terminator ligated into the domestication vector, and so ready for the next level of assembly. In our specific case, sticky ends of the parts are predesigned so that when cleaving both the basic part and the domestication vector BBa_P10500 with BsmBI, the basic piece is ligated and standard overhangs, characteristics for each type of basic piece, are ready for the next level of assembly.

This BBa_P10500 plasmid has a chloramphenicol resistance and the lacZ cassette, so that blue-white screening is performed to select recombinant colonies among the transformed E. coli cells.

This can be thought as Printeria’s PAST.

Figure 1: P10500 domestication vector. Yellow and black puzzle-like pieces represent the restriction sites for BsmbI. It has chloramphenicol resistance.

Figure 2: 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 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. This is what Printeria can assemble nowadays.

As said before, each of these domesticated parts now has a BsaI recognition site and a cleaving 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 end and with RBSs using their right end. At the same time CDSs will stick to these RBSs using their left sticky end, and to the terminators with their right end. Finally, the terminators will stick to the right end of our backbone destination vector so that, we will end up having a plasmid with a single TU inside it.

This is the PRESENT.

Figure 7: pGreen alpha 1 destination vector. The BsmbI restriction site will allow us to create a level 2 assembly.

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) for a level 2 assembly.

Level 2 Assembly

This is the last level of complexity in which, by using a combination of 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 so that its possibilities and combinations are 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