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

Biological Design: The Golden Braid Assembly

We are continuously talking about 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.

Printeria is using a state-of-the-art technology based on the Golden Gate Assembly, the Golden Braid Assembly Method. This technology uses type IIs restriction enzymes in order to cut all the parts and build these genetic circuits.

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 of their recognition sequence. Thus, digestion leaves short single stranded overhangs with non-specific sequences.

This allows us to design the cleaving region so that we are creating a sticky end that will be pasted with the following part, and so on. This is the way in which directionality is maintained and parts are pasted in the desired order.

But why is this assembly technique so crucial for our machine to work?

• 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 so that the whole assembly is taking place in a single step. This fact makes the Golden Braid Technology perfect for our machine to work, as the whole reaction should take place in a single droplet.

• High efficiency. By means of modifying the different parameters we can end up with an almost 100% efficiency.

• Robust reaction. The moving of the droplet across the PCB surface should not be a real problem for it to work.

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

• No scars are left when assembling the different parts.

The Golden Braid Assembly

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 can mainly be divided into three different complexity levels:

Level 0 Assembly

This is the easiest 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 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 when cleaving our domestication vector pUD2 with BsmBI, they are pasted in a proper way.

This pUD2 plasmid has a chloramphenicol resistance and the lacZ cassette so that blue-white screening can be performed 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