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?
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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.
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High efficiency. By means of modifying the different parameters we can end up with an almost 100% efficiency.
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Robust reaction. The moving of the droplet across the PCB surface should not be a real problem for it to work.
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The ability of cutting and pasting several parts by using the same enzymes makes the whole assembly easier to perform.
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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.
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Alberto Conejero lerolerolero
I am member of the Instituto Universitario de Matemática Pura y Aplicada of the UPV. I am also interested in Biomedical Data Analysis, Graph Theory, Network Science, and in the applications of Mathematics to Computational, Systems and Synthetic Biology, and Communication Networks.
I am the author of more than 50 research articles published in international research journals. In addition, I have stayed at the following universities for short periods: in Bowling Green (OH) and Kent (OH) (USA), Lecce (Italy), Prague (Czech Rep.) And Tübingen (Germany).
Before being Director of the Department of Applied Mathematics, I held the position of Director of Academic Performance and Curricular Students Assessment Area of the Vice-rectorate of Students and Culture of the UPV. Previously I held these positions university: Deputy Dean of the ETSINF (formerly Faculty of Informatics) (2004-2009), and Secretary of the Commission of the Strategic Plan of the UPV for the period 2007-2014 (2005-2007).
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