Biosensor library building
ARIA wet lab part development was initially based on two independent but comparable approaches: In vitro approach and the In vivo-In vitro approach. The first one has the goal to prove that our biosensors function by utilizing a commercial LbCas12a coupled with in vitro transcribed gRNAs (using our cloned gRNA plasmids as a DNA template). The latter aims to test if the biosensor-producing machinery can be implemented on living E. coli (in vivo), to then be lysed and used for CRISPR-Cas detection assay (in vitro).
Our initial idea was developing both approaches because CRISPR technology had never been applied as we aimed to do in our innovative In vivo-In vitro approach. However, as experiments went well and we trusted our idea, we only made a few tests of the already tested and traditional in vitro approach so that we had more time to center in our real idea development. Our project has been focused on building the In vivo-In vitro approach, as it confers flexibility and makes possible having self-growable biosensors ensuring availability and reducing production costs.
Alexandria: ARIA's biosensor library
Rapid and accessible nucleic acid detection for clinical diagnosis is paramount for saving time and resources in order to fight against fast pathogens multi-resistant advancement. For this reason, our main goal was to develop a self-growing system that can be distributed worldwide to allow antibiotic resistant bacteria detection and characterization. This system will consist of a library of specific biosensors which we called Alexandria.
Recently, innovative CRISPR-Cas technologies for in vitro diagnosis have been developed as fast tests . So, taking those as a reference we designed different E.coli models, each cloned with a LbCas12a plasmid and/or a gRNA expressing plasmid for antibiotic resistance gene detection. The reporter strategy of our system is based on fluorescence, in a way that each of our biosensors shows luminescence in presence of a specific resistant gene.
As a proof of concept, we have constructed a library of 10 different biosensors for targeting 5 resistance genes available in the lab. As part of the engineering cycle, we had to iterate on the gRNA design in order to achieve the most efficient configuration. That is why we had to rebuild our biosensors and thus have 10 instead of 5 for targeting the same resistant genes. Moreover, future work will consist of constructing biosensors for real application based on our Alpha software data with our already optimized gRNA design.
For the construction of Alexandria (biosensor library), firstly one has to learn how to write and create a book (biosensor) to then replicate the process and obtain the complete collection.
To give a brief description of the biosensor building process, first we have to take a step back and remember the importance of designing the adequate gRNAs for specific target DNA detection. Then, when an optimal design is achieved, the constructs are ordered as ssDNA oligonucleotides that will be pairwise annealed for their cloning into the chosen plasmid backbone. Before this process is carried out, the plasmids aiming to be used may need some trimming or modifications.
Once the desired plasmids with the gRNA sequences are built, they are transformed into the chosen bacterial strain. The bacterial cells can also be co-transformed with another plasmid coding for LbCas12a, or instead two different cells can be used to avoid co-transformation. Then, plating and selection with the corresponding antibiotics are needed prior to culture and stock preparation.
Come along and keep reading for more insight into the creation of the Alexandria library…
A standard Polymerase Chain Reaction (PCR) is an in vitro method that allows a single, short region of a DNA molecule to be copied multiple times by Taq Polymerase .
The strategic design of the primers allows some retouching to the DNA fragment wanting to be amplified, such as deletion or modification of specific sequences.
Once the PCR has been carried out it is necessary to digest the DNA template, which is the initial plasmid. In our case, a simple incubation with a DpnI restriction enzyme was performed. This nuclease is specific for methylated and hemimethylated DNA . Since the plasmids were isolated from dam+ E. coli, they will have methylated adenines in any GATC sequence, thus will be digested by DpnI .
To conclude with the plasmid modification protocols, a final ligation of the PCR product must be performed. Previous to ligation, phosphorylation is needed for adding phosphate groups at the extremes of the linear DNA, to then be able to join them and obtain circular DNA. This reaction can be achieved thanks to the T4 Polynucleotide Kinase (PNK), since it transfers the γ-phosphate from ATP to the 5´ end of nucleotides . Then, incubating with T4 DNA ligase will be the final step for joining the linear PCR product.
In order to keep a stock of these modified plasmids, it is necessary to transform them into NYZ5α competent E. coli (similar properties to DH5α). This bacterial strain is known for having specific features suitable with high-efficiency plasmid transformation. It has a recA mutation, so it will not be able to perform heterologous recombination, ensuring a higher insert stability. Also, NZY5α lack on some endonucleases, which could digest the plasmid during isolation procedures . Transformation of this bacterial strain requires a heat-shock protocol .
Plating for transformed cells selection
Plating of the transformed cells will let them grow in a favorable environment . Also, the addition of antibiotic to the media is key to select the cells that have incorporated the plasmid. Since these will have an antibiotic resistance gene, provided by the introduced plasmid, they will survive and grow in single colonies.
This experiment aims to verify if the grown colonies have correctly incorporated the modified plasmids. After the PCR has been performed for a few of the colonies, electrophoresis on an agarose gel will reveal the presence or absence of the PCR amplicon and the size of the product .
This technique allows sequence verification of important plasmid features, in our case MBP deletion or insertion of a timine in the direct repeat sequence. Consists of selecting one or more unique oligonucleotide primers that flank the regions of your plasmid that you wish to confirm .
Once the backbone plasmids are assembled and transformed into NYZ5α cells, it is necessary to prepare the DNA inserts that will be cloned into them. In this case, these will be pairs of complementary ssDNA oligonucleotides codifying for each gRNA. Furthermore, we have to take into account that the insert will have to bind to the cleaved plasmid backbone. Consequently, the DNA sequence must include overhangs complementary to the sticky ends that will be formed during the assembly.
Once the ssDNA oligos have been ordered and synthesized, they can go through an annealing protocol  which will result in a dsDNA fragment ready to be cloned.
Golden Gate assembly is a quite useful cloning method that relies on Type II restriction enzymes. These have the particularity of cleaving outside of the recognition site. As a result, these sites are eliminated by digestion/ligation and do not appear in the final construct . Particularly, our gRNA backbone plasmids were designed to be digested with BsaI endonuclease.
Furthermore, Golden Gate cloning allows performing a single 30-minute reaction where digestion and ligation are taking place simultaneously .
Again, the cloned plasmids must be transformed into the corresponding E. coli strain. This time, the main interest of the transformed cells will be to express high amounts of the heterologous gene. The ideal strain for performing this job would be BL21, due to its lack of Lon protease and OmpT membrane protease . Transformation of this bacterial strain can be carried out via electroporation, where an electrical pulse induces the formation of transient membrane pores .
Plating of the transformed cells will let them grow in a favorable environment . Also, the addition of antibiotics to the media is key to select the cells that have incorporated the plasmid. Since these will have an antibiotic resistance gene, provided by the introduced plasmid, they will survive and grow in single colonies.
BL21 E. coli cells have been transfected with lambda DE3 prophage, thus they contain the T7 RNA polymerase gene. Its expression is regulated by the lacUV5 promoter, being inducible by the presence of IPTG . When this molecule is added to the liquid bacterial culture, BL21 cells can induce expression of this polymerase. Consequently, T7 polymerase will transcribe the plasmids’ heterologous genes, since they are under the regulation of the constitutive T7 promoter.
This is how we induce the synthesis of our biosensors’ main components: LbCas12a and gRNA.
The last engineering cycle stage is testing the built biosensors. See details about the testing procedure in the Biosensor library testing page.
Gibson Assembly Cloning
Gibson Assembly is another efficient cloning method usually used for the assembly of multiple fragments. This method takes advantage of adjacent DNA fragments that have complementary ends, to fuse two different parts together .
For this reason, to clone the DNA fragment coding for the autolysis protein (protein E) it is necessary to first add overhangs of about 15-20 bp with the complementary sequence of the adjacent regions of the site where it is to be inserted. This was done by PCR amplification, whose primers should contain the sequence of the overhang and part of the sequence of the fragment to be cloned.
In parallel, the plasmid with the PBAD promoter and the chloramphenicol resistance is the one that will be used as a backbone for the insertion of the protein. Therefore, it must be digested by two restriction enzymes, NheI and PstI, to open it.
After purifying all the necessary parts, the same volume of the parts is added as the Master Mix for Gibson Assembly at 2x concentration. This mix contains an exonuclease that creates single-stranded overhangs that will bind by complementarity, a DNA polymerase fills in the gaps created, and a ligase joins all the fragments together. This entire reaction is carried out at 50°C for one hour .
Plasmids transformation into NZY5α competent cells
In order to induce the autolysis of cells, the plasmid needs to be transformed into them. The steps to follow are described in the "Transformation of the modified plasmids into NYZ5α competent cells" section above.
However, it should be noted that when such cells are to be grown on Agar plates, they must contain a high concentration of glucose that can inhibit the leakiness of the pBAD promoter. The contrary situation can be found described in the Results page .
Cells clean-up and induction
Once colonies that have successfully transformed have been picked and left to grow in a liquid culture with D-Glucose, these cells have to be cleaned. They are first centrifuged to form a pellet, then the supernatant containing glucose is removed and finally, a new clean culture medium is added.In the last step, autolysis protein expression is induced by L-Arabinose. To read the induction procedure go to Biosensor Library Testing Page
 Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345 doi: 10.1038/nmeth.1318
 Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 177(14), 4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995