Biological Design Challenges
As we have explained in our result page , when developing Printeria, we need to optimize the largest number of variables in order to achieve the highest degree of automation.
This is what we have tried to achieve with our Biological Design:
An assembly method that allows easy automation with sufficient robustness for the reaction to be carried out even under suboptimal conditions.
A standard set of DNA parts for that assembly method
A way to avoid plate screening to differentiate transformed bacteria with the vector containing the correct insert from those that do not have the insert, as this is the step that most complicates automation.
A way to introduce competent E. coli that avoids us having to keep them deep-frozen
How did we design the biological part of Printeria to get all this?
First of all, the assembly method chosen is the Golden Gate assembly method . We have chosen this technology for several reasons:
This technology uses type IIS restriction enzymes in order to cut all the parts and build these genetic circuits. These 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 method 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.
Shorter scars are left when assembling the different parts. If the overhangs are carefully designed, scarless DNA junctions can be obtained.
The way to avoid the plate screening step is to use target plasmids previously digested with the endonuclease used in level 1 assemblies.Using the vectors in their linearized form, the number of transformations with vectors without the insert is greatly reduced, allowing cultures to be obtained in liquid medium expressing the protein of interest without the need to pass through the plate. However, the culture obtained will not be pure, and could only be obtained by isolating colonies (for example, with a triple streak).
With regard to the way of entry of not deepfrozen competent E.coli, we have chosen electrocompetents stored at -20ºC. The reasons are as follows:
Electroporation is the most efficient method of transformation.
We checked that the electrocompetent cells keep the competence stored at -20ºC. You can check the data in experiments.
The implementation in the hardware was very simple because the electroporator was already designed by the Valencia UPV iGEM team for the Hype It project.
The Golden Braid Assembly method
GoldenBraid method relies on Golden Gate Technology for the assembly of Composite part and introduces a double-loop strategy for the assembly of multigenic constructs. In the Golden Gate assembly method the Composite Parts can be combined in binary steps to create multigene structures (several Composite Parts 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 Golden Gate can be divided into three different steps:
Level 0 Assembly
This is the Golden Gate reaction needed for the adaptation of any DNA sequence to the Golden Gate standard. It implies the removal of internal restriction sites for the enzymes used in Golden Braid (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 (adaptation to the Golden Braid 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 composite part. 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: Basic domesticated part inside the BBa_P10500. 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 5: 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 composite part.
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 Composite Parts 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 Golden Braid Level 2 (Figure 11).
Figure 6: pGreen alpha 1 destination vector. The puzzle-like pieces represent the restriction sites for BsaI. It has kanamycin resistance.
Figure 7: BsaI digested destination and domesticated part to build a composite part.
Figure 8: Composite part insertion inside pGreen alpha1
Figure 9: Light coloured sequences represent the BsaI sticky ends which have been ligated. 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 composite parts 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.
Figure 10: Composite Part ready to be used in a Level 2 assembly.
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