Team:UPF Barcelona/Wetware building

ARIA

Wetware

Biosensor library building

Building the biosensors is the second engineering cycle stage. Here, the main steps followed for the building process are exposed.

Approach definition

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 [1]. 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.

In this image, the main architecture of ARIA’s synbio system is shown. Our specific biosensors enable fluorescence once activated, and they are placed on a paper-based structure.

Figure 1: Basic Alexandria biosensor library schematic.

Experiments replication

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…

Plasmids building

PCR amplification of the desired backbone sequence

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 [2].

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.

In this image it is shown how one dsDNA chain is amplified by PCR to eight dsDNA chains. First, the dsDNA is denatured, at 94ºC, forming two ssDNA chains. After this step, ssDNA chains are annealed with it’s complementary primer and the synthesis reaction is performed at 72ºC. Finally, eight dsDNA chains are synthesized.

Figure 2: PCR Reaction.

We used this technique to amplify our plasmids excluding the MBP coding region of the pMBP-LbCas12 plasmid. Also, we added a T (timine) nucleotide into the direct repeat (DR) sequence of our gRNA plasmid (for more details visit project design page).

Protocol

Digestion of the template plasmid

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 [3]. Since the plasmids were isolated from dam+ E. coli, they will have methylated adenines in any GATC sequence, thus will be digested by DpnI [4].

Protocol

This picture shows how the DpnI digests the circular dsDNA template on the methylated/hemimethylated site.

Figure 3: Digestion of the PCR DNA template

Phosphorylation of the amplified plasmid sequence and ligation

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 [5]. Then, incubating with T4 DNA ligase will be the final step for joining the linear PCR product.

Protocol

This picture represents at the left part 2 dsDNA chains that are united to non complementary nucleotides. Then, the DNA is submitted to heat and the 2 ssDNA chains are obtained. Finally, they are cool and the 2 ssDNA chains are annealed forming a dsDNA chain totally complementary.

Figure 4: Ligation of the linear PCR product

Transformation of the modified plasmids into NYZ5α competent cells

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 [6]. Transformation of this bacterial strain requires a heat-shock protocol [7].

Protocol

This image shows in first place an eppendorf tube, that is on ice, where we have the competent cells. In second place, shows the incubation of the competent cells with the plasmid DNA on ice. In third place, the mentioned tube is introduced in a water bath at 42 ºC during 30” (Heat shock) and here the plasmids are incorporated inside the competent cells. Next, they are transferred again to ice and we finally obtain the transformed cells.

Figure 5: Heat Shock Transformation

Post-transformation steps:

Plating for transformed cells selection

Plating of the transformed cells will let them grow in a favorable environment [8]. 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 picture shows how LB-agar and the specific antibiotic are mixed and then put on a petri plate. Next, this plate is solidified and ready for use.

Figure 6: Pour Plate Method

In this image, the procedure for plate culturing is represented. The first step shown is loop sterilization in the flame. Next, it’s picking liquid cell culture with the loop for plate inoculation. Inoculation is done in three steps: firstly, drawing parallel lines with the loop until half of the plate is inoculated; then, changing the direction of the strokes, a quarter of the plate has to be covered; finally, changing the direction again, the remaining space of the plate must be traced with what’s left on the loop.

Figure 7: Plate culture

Colony PCR

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 [9].

Protocol

In this image, there is represented on the left side a plasmid marked with a target sequence. This target sequence is flanked by two primers that will amplify the region of interest. Next, the primers, the plasmid, and the polymerase (that are necessary for the PCR reaction) are displayed. Finally, the results of the PCR are revealed in a gel for its analysis.

Figure 8: Colony PCR Set-up and Analysis

Sequencing

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 [10].

Protocol

On the top left, a graphic drawing of a spectrophotometer is shown. This instrument facilitates an absorbance measurement of the DNA material aiming to be sequenced. Next, a mix of the primer, DNA material and Milli-Q water is done in an Eppendorf tube. Then, the tube is labeled and sent to the sequencing service. The final step shown is a graphic that sums up the sequencing results.

Figure 9: PCR product Sequencing and Analysis.

Transformed NZY5α E. coli liquid culture

Once the bacterial cells have been transformed, we will want to obtain a high-density bacterial culture. This way a greater number of cells are grown, being more feasible to isolate enough plasmid for experimental use [11].

Protocol

The image shows the preparation of liquid culture. First, LB media is added into a 15mL tube, next is mixed with the specific antibiotic. Then, the 15mL preparation is inoculated from a cell culture plate with a sterile loop, and finally, it’s all incubated at 37ºC.

Figure 10: Liquid Culture Preparation.

Bacterial Glycerol Stock

It is important for long-term storage of plasmids. This way, we keep a stock of the modified backbone plasmids. This saves us from having to prepare more competent cells and transform them again [12].

Protocol

This image represents the preparation of a glycerol stock, adding both liquid culture and glycerol 50% solution in equipable quantities. It is all added directly to a cryotube.

Figure 11: Glycerol Stock Preparation.

Pairwise annealing of the ssDNA oligos (gRNA spacers)

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 [13] which will result in a dsDNA fragment ready to be cloned.

Protocol

This picture represents an annealing protocol for partially complementary ssDNA oligonucleotides.

Figure 12: Annealing procedure.

Golden Gate assembly for cloning the spacer sequence into the modified gRNA plasmid

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 [14]. 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 [14].

Protocol

A scheme of Golden Gate assembly is represented in various steps. In the first place, the backbone plasmid is cleaved in the restriction sites by BsaI. Then, the DNA insert with complementary overhangs to the backbone sticky ends will bind to it. Next, the DNA ligase will unite the gene of interest and the DNA backbone, finally generating the cloned vector.

Figure 13: Cloning with Golden Gate Assembly.

Transformation of the gRNA and LbCas12a plasmids into BL21 competent cells

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 [15]. Transformation of this bacterial strain can be carried out via electroporation, where an electrical pulse induces the formation of transient membrane pores [16].

Protocol

This image shows in the first place an Eppendorf tube, that is on ice, where we have the competent cells. In the second place, shows the incubation in a cuvette, of the competent cells with the plasmid DNA on ice. In third place, we can observe how the cuvette is subjected to an electric shock and how the plasmids are being incorporated into the competent cells. Next, they are transferred again to ice. Finally, cells have been transformed.

Figure 14: Electroporation cell transformation.

Plating for selection

Plating of the transformed cells will let them grow in a favorable environment [8]. 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.

In this image, the procedure for plate culturing is represented. The first step shown is loop sterilization in the flame. Next, it’s picking liquid cell culture with the loop for plate inoculation. Inoculation is done in three steps: firstly, drawing parallel lines with the loop until half of the plate is inoculated; then, changing the direction of the strokes, a quarter of the plate has to be covered; finally, changing the direction again, the remaining space of the plate must be traced with what’s left on the loop.

Figure 15: Plate Culture.

Colony PCR

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 [9].

Protocol

In this image, there is represented on the left side a plasmid marked with a target sequence. This target sequence is flanked by two primers that will amplify the region of interest. Next, the primers, the plasmid, and the polymerase (that are necessary for the PCR reaction) are displayed. Finally, the results of the PCR are revealed in a gel for its analysis.

Figure 16: Colony PCR Set-up and Analysis

Sequencing of the insert flanking region

This technique allows sequence verification of important plasmid features, in this case it will cover the region flanking the DNA insert. Consists of selecting one or more unique oligonucleotide primers that flank the regions you wish to confirm [10].

Protocol

On the top left, a graphic drawing of a spectrophotometer is shown. This instrument facilitates an absorbance measurement of the DNA material aiming to be sequenced. Next, a mix of the primer, DNA material and Milli-Q water is done in an Eppendorf tube. Then, the tube is labeled and sent to the sequencing service. The final step shown is a graphic that sums up the sequencing results.

Figure 17: PCR product Sequencing and Analysis.

Liquid culture

Once the bacterial cells have been transformed, we will want to obtain a high-density bacterial culture. This way a greater number of cells are grown, being more feasible to isolate enough plasmid for experimental use [11].

Protocol

The image shows the preparation of liquid culture. First, LB media is added into a 15mL tube, next is mixed with the specific antibiotic. Then, the 15mL preparation is inoculated from a cell culture plate with a sterile loop, and finally, it’s all incubated at 37ºC.

Figure 18: Liquid Culture Preparation.

Glycerol stock

It is important for long-term storage of plasmids. This way, when the desired biosensors want to be isolated, the respective coding plasmids will already be in the desired bacterial strain. This saves us from having to prepare more competent cells and transform them again [12].

Protocol

This image represents the preparation of a glycerol stock, adding both liquid culture and glycerol 50% solution in equipable quantities. It is all added directly to a cryotube.

Figure 19: Glycerol Stock Preparation.

Expression of the gRNA and Cas12a plasmids under IPTG induction

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 [15]. 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.

Protocol

Biosensor library testing

The last engineering cycle stage is testing the built biosensors. See details about the testing procedure in the Biosensor library testing page.

Autolysis complex building

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 [17].

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.

Protocol

Shown is the preparation of the fragments prior to cloning with Gibson Assembly. On the one hand, 15-20bp of overlapping ends are added to the E protein fragment by PCR. On the other hand, the plasmid that will serve as backbone is digested by two restriction enzymes, NheI and PstI.

Figure 20: Fragments preparation for Gibson Assembly.

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 [18].

Protocol

A scheme of the Gibson Assembly is illustrated in several steps. The first step is the creation of 3' overhangs by an exonuclease. The second step is the extension of the free gaps by a DNA polymerase. In the third step a Ligase joins all the cloning fragments together.

Figure 21: Gibson Assembly scheme in several steps.

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 [19].

Protocol

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

Protocol

The image represents in several steps all the cells' clean-up process. First, cells are centrifuged, then supernatant media is removed and a new one is added. Finally, cells are induced with L-arabinose.

Figure 22: Clean-up process and L-arabinose induction.

References

[1] Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F., & Collins, J. J. (2021). CRISPR-based diagnostics. Nature Biomedical Engineering, 5(7), 643–656. doi: 10.1038/s41551-021-00760-7

[2] Polymerase Chain Reaction (PCR).(n.d.). Addgene.org

[3] DpnI(n.d.). Jena Bioscience website

[4] Patrick, M. (2016, June 30). Plasmids 101: Methylation and Restriction Enzymes. Addgene Blog. Retrieved October 6, 2021, from: Addgene. Link here.

[5] T4 Polynucleotide Kinase. (n.d.). Promega. Retrieved October 6, 2021, from:NEB. Link here.

[6] NZY5α. (n.d.). Nzytech. Retrieved October 6, 2021, from:Promega

[7] Bacterial Transformation Workflow–4 Main Steps. (n.d.). ThermoFisher Scientific. Retrieved October 6, 2021, from: Thermofisher

[8] Introduction to Cell Culture - UK. (n.d.). Retrieved from Thermofisher

[9] New England Biolabs. (n.d.). Colony PCR. Retrieved September 26, 2021, from Neb.com website: NEB.

[10] Addgene: Protocol - how to perform sequence analysis. (n.d.). Retrieved September 26, 2021, from: Addgene

[11] Addgene: Protocol - how to inoculate a bacterial culture. (n.d.). Retrieved September 26, 2021, from: Addgene

[12] Addgene: Protocol - how to create a bacterial glycerol stock. (n.d.). Retrieved September 26, 2021, from: Addgene

[13] Annealing Oligonucleotides Protocol. (n.d.). Sigma Aldrich. Retrieved October 6, 2021, from: Sigmaaldrich

[14] Gearing, M. (2015, August 27). Plasmids 101: Golden Gate Cloning. Addgene Blog. Retrieved October 6, 2021, from: Addgene. Link here.

[15] BL21(DE3) Competent Cells. (n.d.). ThermoFisher Scientific. Retrieved October 6, 2021, from: Thermofisher

[16] Electroporation. (n.d.). ThermoFisher Scientific. Retrieved October 6, 2021, from: Thermofisher

[17] 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

[18] New England Biolabs. (n.d.). Gibson Assembly®. Retrieved October 19, 2021. Retrieved from: NEB

[19] 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

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