Results

Overview

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This year our team decided to deal with a project which combines innovative synthetic biology-based methodologies and engineering tools with state-of-the art technologies for 3D-printed microfluidics manufacturing. Knowledge from different scientific fields including molecular biology, biochemistry and nanotechnology was employed. To construct the final DIAS detection platform for accurate target miRNA detection, multiple basic “components” had to be developed from scratch and subsequently efficiently assembled as a functional detection system with high reliability. After conceptualizing the idea behind DIAS system, considerable in silico work had to be done to efficiently design the desire genetic parts for our system. And then comes the lab work. Several PCR methodologies along with multiple cloning procedures were employed to construct the final RFC [10] compatible plasmids bearing the desired sequences for the LbuCas13a expression and the crRNAs in vitro transcription. Afterwards, several experiments were conducted to efficiently express and purify the LbuCas13a protein for subsequent experiments in the detection assay. Regarding the microfluidic chips manufacturing, several trial-and-error methodologies were employed to construct the chip in its final form. And last but not least, the system's "components" produced in the previous experimental steps had to be tested as a "whole functional machine" in several detection assays. Below we provide an extensive description of our experimental results which could be divided into 4 main categories:

• PCR amplification and cloning experiments.
• LbuCas13a protein expression and purification.
• Droplet microfluidic chip manufacturing.
• Feasibility of LbuCas13a/crRNA system for miRNA detection in bulk solution.

This section provides all the necessary experimental results regarding the PCR amplification steps and cloning experiments that we followed to construct the final pSB1C3 plasmid. Further information regarding the cloning strategy that employed for the construction of each plasmid is provided on the experiments page.

Cloning experiments

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Results from cloning experiments

Construction of Cloned SUMO-LbuCas13a Repos (BBa_K4170014) plasmid

This plasmid contains the coding sequence (CDS) of codon-optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) protein fixed to pSB1C3 backbone utilizing two standard techniques: a) mutagenesis PCR amplification; b) Golden Gate Assembly.

Mutagenesis PCR was performed for Cas13a sequence by combining 4 pair of primers, fragmenting LbuCas13a into 4 parts (Cas13a P1, Cas13a P2, Cas13a P3, Cas13a P4), as described in the experiments page. After the completion of the mutagenesis PCR amplifications, 1% agarose gel electrophoresis was conducted. As shown in Figure 1 all the PCR amplifications DNA ladder 100bp succeeded. Cas13a P1 part (1240 bp), Cas13a P2 part (734 bp), Cas13a P3 part (1338 bp), Cas13a P4 part (646 bp) and pSB1C3 backbone linearized (2052 bp) are depicted in Figure 1.

Afterwards, Golden Gate assembly of the PCR amplified Cas13a P1, P2, P3, P4 parts with the linearized pSB1C3 vector was performed for the efficient construction of the SUMO-LbuCas13a coding sequence into PSB1C3 plasmid. The products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells. To verify the successful Golden Gate assembly, colony PCR was performed using the primers VR and VF2. Then the samples were loaded and run in 1 % agarose gel electrophoresis. As depicted on Figure 2 from all colonies the desired SUMO-LbuCas13a (5908 bp) part has been amplified. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20 performing the template DNA of the colony PCR. The other half amount was picked for the overnight liquid culture.

Construction of Cloned final T7-SUMO-LbuCas13a (BBa_K4170016) plasmid

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) under the transcription control of T7 promoter fixed to pSB1C3 backbone. For the construction of this part two standard techniques were utilized: a) PCR amplification; b) Golden Gate assembly.

PCR amplifications were performed utilizing 3 pairs of primers and then 1 % agarose gel electrophoresis was conducted. As shown in Figures 3 and 4 all the PCR amplifications succeeded. In Figure 3 T7-Cas13a P1 part (1261 bp) and T7- Cas13a P0 part (1560 bp) are depicted. In Figure 4 T7-Cas13a P2-P4 part (2678 bp) is depicted. The linearized pSB1C3 backbone was not amplified again, as the amount of its first amplification was enough for all the experiments that followed (Figure 1).

Afterwards, Golden Gate assembly of the PCR amplified T7-Cas13a P0, P1, P2-P4 parts with the linearized pSB1C3 vector was performed for the efficient construction of the fina T7-SUMO-LbuCas13a plasmid. The products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using two pairs of primers: VR / VF2 and T7-Cas13a-P0-FWD / Cas13a-P4-RVS. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture. The samples were loaded and run 1 % agarose gel electrophoresis we concluded that the Golden Gate assembly was successful. As depicted in Figure 5 the outcomes of the colony PCR (VR/VF2) five out of six colonies have received the desirable insert (5713 bp). The next five samples are the outcomes of the colony PCR (T7-Cas13a-P0-FWD / Cas13a-P4-RVS) from 5 different colonies. All the colonies have received the desired insert (5446 bp).

Construction of Cloned T7-LbuCas13a-SUMOLESS BBa_K4170017

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized LbuCas13a protein without the small ubiquitin-like modifier (SUMO) protein fixed to pSB1C3 backbone utilizing two standard techniques: a) PCR amplification; b) Golden Gate Assembly.

PCR amplification was performed utilizing two pairs of primers and then the samples were loaded and run in 1 % agarose gel electrophoresis. As shown in Figure 6 both PCR amplifications succeeded. On the left side the SUMOLESS-Cas13a part (3521 bp) is depicted and on the right side SUMOLESS-T7 part (1640 bp). The linearized pSB1C3 backbone was not amplified again, as the amount of its first amplification was enough for all the experiments that followed (Figure 1).

Afterwards, Golden Gate assembly of the PCR amplified SUMOLESS-Cas13a and SUMOLESS-T7 parts with the linearized pSB1C3 vector was performed for the efficient construction of the T7-LbuCas13a-SUMOLESS plasmid. The products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using VR and VF2 primers. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 20 μl of dH20. The other half amount was picked for liquid culture. The samples were loaded and run in 1% agarose gel electrophoresis and then we concluded that the Golden Gate assembly was successful. As depicted in Figure 7 from all colonies the SUMOLESS Cas13a (5395 bp) has been amplified.

Construction of Cloned Rhamnose Repos BBa_K4170054

This plasmid contains the rhaBAD operon and rhaR-rhaS activators of E.coli flanked by BioBrick Preffix and Suffix regions into the PSBIC3 backbone. For the composition of this part two standard techniques were utilized: a) colony PCR from DH5a competent E.coli cells; b)Golden Gate Assembly.

After the completion of the colony PCR amplifications, 1 % agarose gel electrophoresis was conducted. As shown in Figure 8 Rha (2091 bp) from colonies 1 and 3 were slightly amplified while amplification from colony 2 was unsuccessful. For our next experiments we used RhaBAD-rhaR-rhaS colony part amplified from colony 4 which was the most successfully amplified colony.

Afterwards, Golden Gate assembly of the colony PCR amplified rhaBAD-operon part with the linearized pSB1C3 vector was performed for the efficient construction of the Rhamnose Repos plasmid. The products of the Golden Gate assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using VR and VF2 primers. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture. Picking samples from different colonies and then evaluating the results on a 1% agarose gel electrophoresis, we concluded that the Golden Gate assembly was successful on colonies 3 and 4. As depicted on Figure 9 from 3 and 4 colonies the Rhamnose-Repos (2345 bp) has been amplified, while from 1 and 2 colonies has not.

Construction of Cloned Rhamnose-SUMO-LbuCas13a BBa_K4170055

This plasmid contains all the basic genetic parts required for the bacterial expression of codon-optimized LbuCas13a protein with the small ubiquitin-like modifier (SUMO) protein under the transcriptional control of rhaBAD promoter regulated by rhaS and rhaR activators into PSBIC3 backbone. For the composition of this part two standard techniques were utilized: a)PCR amplification; b) Golden Gate assembly.

After the completion of the PCR amplifications, 1 % agarose gel electrophoresis was conducted. As shown in Figure 10 both PCR amplifications succeeded. On the left side the rhaR-rhaS part (2119 bp) is depicted and on the right side Cas13a-RBS-P4 RVS part (3903 bp).

Final T7-SUMO-LbuCas13a diagnostic digestions

After the cloning of plasmid final T7-SUMO-LbuCas13a, that contains all the basic genetic parts required for the bacterial expression of codon-optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) protein, diagnostic digestions followed, in order to verify with a different method than colony PCR that the cloning was successful. We performed a double digestion using the restriction enzymes EcoRI and SpeI and a single digestion using the restriction enzyme EcoRI. Then a 1% agarose gel electrophoresis was conducted. From the double digestion two bands should arise (5422 bp and 2047 bp), while from the simple digestion with EcoRI only a 7469bp band should arise. From this experiment final T7-SUMO-LbuCas13a plasmids that have been isolated from 6 different colonies were used. As depicted in Figure 11 all the digestions were successful.

Construction of Cloned crRNAs

For the purposes of this project, we conducted the cloning of six crRNAs: crRNA-3ps, BBa_K4170019, crRNA-3pm, BBa_K4170021, crRNA-3pe, BBa_K4170022, crRNA-5ps, BBa_K4170042, crRNA-5pm, BBa_K4170043, crRNA-5pe.BBa_K4170044. crRNA is a fundamental part of CRISPR/Cas13a system and its structure consists of a stem-loop supporting the Cas13a/crRNA duplex and a spacer performing the guidance of the system.

• crRNA-3ps targets the miRNA-17-3p since it contains the nucleotide sequence for the perfect hybridization with the mature miRNA-17-3p.
• crRNA-3pm consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence with a mismatch \( (T \to A) \) in position 14 generating no perfect hybridization of the spacer with the mature miRNA-17-3p.
• crRNA-3pe consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence containing an extra hairpin loop in the 3' end.
• crRNA-5ps targets the miRNA-17-5p since it contains the nucleotide sequence for the perfect hybridization with the mature miRNA-17-5p.
• crRNA-5pm consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence with a mismatch \( (C \to G) \) in position 14 generating no perfect hybridization of the spacer with the mature miRNA-17-5p.
• crRNA-5pe consists of the loop sequence for the construction of Cas13a/crRNA duplex and a spacer sequence containing an extra hairpin loop in the 5' end.

Each plasmid was constructed with direct PCR amplification of PSB1C3 plasmid utilizing 2 pairs of primers followed by a Golden Gate-based "SevaBrick Assembly" method. The first pair consists of a primer (loop RVS standard) binding to the Biobrick prefix sequence containing an overhang with the stem-loop sequence of the crRNA and a primer (loop FWD standard) binding to the backbone of the plasmid. The second pair of primers consists of a primer (crRNA spacer FWD interchangeable) binding to the Biobrick suffix sequence containing an overhang with the spacer sequence of the crRNA and a primer (spacer RVS standard) binding to the backbone of the plasmid. This strategy enables the handy and fast alternation of the overhang sequence of the interchangeable primer for the construction of any crRNA flanked in PSB1C3 plasmid keeping the other primers as customary components. Each plasmid contains all the basic genetic parts required for the in vitro transcription of the different crRNAs regulated by T7 promoter.

After the completion of the PCR amplifications, 1% agarose gel electrophoresis was conducted. The samples also contain DNA templates complementary to the crRNA molecules. According to Figure 12 all the PCR amplifications succeeded, using the appropriate primers as shown on the above table. Loop part (1209 bp) and 3ps, 3pm, 3pe, 5ps, 5pm, 5pe spacer parts are depicted.

Then, Golden Gate assembly of the PCR amplified loop part and each crRNA-spacer part was conducted, for the efficient construction of the crRNA coding sequence under the transcriptional control of the T7 promoter. The Golden Gate assembly products underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using the primers VR and VF2. Picking sample from different colonies and then evaluating the results on a 1 % agarose gel electrophoresis we concluded that the Golden Gate assembly was successful. As depicted on Figure 13 from all colonies has been amplified the desired inserts. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture.

Digestions of the cloned crRNA plasmids

For further assurance that the DNA templates complementary to the crRNAs have been inserted to the pSB1C3 plasmid, diagnostic digestions followed. As depicted in Figure 14 a double enzymatic digestion by XbaI and PstI restriction enzymes indicatively was performed for the cloned crRNA 3pm (102 bp) and cloned crRNA 5pm (102 bp).

In vitro transcription of crRNAs

For the in vitro transcription of crRNAs the plasmids were linearized utilizing the enzyme SapI which recognized the restriction site we had insert in the plasmid downstream of the crRNA sequence.

The linearized DNA template bands were purified by Nuclospin Gel and PCR Clean-up kit (Macherey-Nagel, Duren, Germany) and a second purification step with phenol/chloroform was performed. The next step was the template-directed synthesis of the crRNAs through in vitro transcription using T7 Polymerase (HiScribe T7 High Yield RNA Synthesis Kit, NEB) and the purification of the products, using Monarch RNA Cleanup Kit (NEB). In Figure 16 the crRNA transcripts are depicted on a 3% agarose gel electrophoresis after in vitro transcription, while in Figure 17, crRNA transcripts are depicted after the clean-up procedure. In both Figures, the samples were loaded with the following order: crRNA-3p standard design (59bp), crRNA-3p mismatch design (59bp), crRNA-3p extra-loop (64bp), crRNA-5p standard design (59bp), crRNA-5p mismatch design (59bp), crRNA-5p extra-loop design (64bp)

Construction of Cloned LbuCas13a-SUMO with crRNAs

In our effort to synthesize the ribonucleoprotein SUMO-LbuCas13a-crRNA for the 3pm and 5pm crRNAs, there were constructed two plasmids. The first one contains all the basic genetic parts required for the bacterial transcription of crRNA 3pm and for expression of codon optimized LbuCas13a protein in fusion with small ubiquitin-like modifier (SUMO) under the transcription control of T7 promoter fixed to pSB1C3 backbone. The second one consists of the same parts, but the sequence for the bacterial transcription of crRNA 5p instead. For the construction of these plasmids for the ribonucleoprotein expression, two different methods were applied: a) Golden Gate assembly and b) 3A Assembly.

Golden Gate Assembly method

Firstly, PCR amplifications were performed utilizing 3 pairs of primers and then 1 % agarose gel electrophoresis was conducted. As shown in Figures 18-19 all the PCR amplifications succeeded. In Figure 18 the SUMO-Cas13a for ribonucleoprotein (5481 bp) is depicted. In Figure 19, crRNA 3p for ribonucleoprotein (152 bp), crRNA 5p for ribonucleoprotein (152 bp) and the pSB1C3 backbone linearized (2052 bp) are depicted. Afterwards, Golden Gate assembly of the PCR amplified products was performed for the efficient construction of the SUMO-LbuCas13a-crRNAs coding sequence into PSB1C3 plasmid. The products of the Golden Gate assembly underwent transformation into E. coli DH5a competent cells. To verify the successful Golden Gate assembly, colony PCR was performed using the primers VR and VF2. Then the samples were loaded and run in 1 % agarose gel electrophoresis, but from none colony the desired SUMO-LbuCas13a-crRNA (5797bp) part has been amplified.

3A Assembly method

Firstly, 3 double digestions were performed utilizing 3 pairs of restriction enzymes, in order to create the two parts that will be assembled and the linearized backbone plasmid. Then 1% agarose gel electrophoresis was conducted to evaluate the results. As shown in Figures 20 and 21, all the digestions succeeded. In Figure 20 the double digestion of cloned crRNA-3ps, 3pm, 3pe plasmids and cloned crRNA-5pm, 5pm, 5pe plasmids with XbaI and PstI restriction enzymes are depicted. From the two bands (102 bp and 2044 bp) that aroused from both digestions, 102 bp part was used on the next steps. In Figure 21 the double digestion of final T7-SUMO-Lbu Cas13a and pSB1C3 plasmid are depicted. Final T7-SUMO-LbuCas13a was digested with EcoRI and SpeI restriction enzymes and two parts aroused (2047 bp and 5422 bp). The 5422 bp part was utilized in the next step. pSB1C3 plasmid was digested with EcoRI and PstI restriction enzymes and the 2047bp linearized part will be used in the next step. As long as the three assembling parts were created with the appropriate ends, ligation protocol followed. The products of the 3A assembly underwent transformation into E.coli DH5a competent cells. To verify the successful 3A assembly, colony PCR was performed using the VR and VF2 primers. Then, the samples were loaded and run in 1% agarose gel electrophoresis. As depicted on Figure 22 from all colonies, except colonies, the desired SUMO-LbuCas13a-crRNA part has been amplified. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10μl of dH2O performing the template DNA of the colony PCR. The other half amount was picked for the overnight liquid culture.

Protein experiments

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Results from SUMO-LbuCas13a expression and purification

LbuCas13a protein expression procedure with 4 hours induction, at 37 oC using 1mM IPTG

Initially, a four-hour induction of 1L bacterial culture, at 37oC with addition of high IPTG induction factor, of 1mM was tested. The aim was to observe the behavior of the system and decide the course of action. 1ml of the bacterial culture had been removed every hour (creation of timeline), and when the induction ended, the soluble part of SUMO- LbuCas13a was separated from the insoluble part, according to the protocol, as described in the experiments page. The samples then were analyzed using 8% gel electrophoresis, and an immunoblotting method was performed (Western Blot technique), using an Anti-His primary antibody (Santa Cruz Biotechnology) in 1:2000 dilution ratio.

Based on the visual observation of the sample analysis we concluded that, the use of high temperature and high concentration of IPTG, were not enough to promote the protein expression in the soluble part. Therefore, the SUMO booster sequence could not function during the 4h induction. In contrast to the above, the insoluble part (inclusion bodies) was observed to contain the protein of interest, but not in a quantity capable enough to be exploited in later procedures.

LbuCas13a protein expression procedure with overnight induction, at 25oC using 1mM IPTG

After the first inefficient attempt of the LbuCas13a protein expression, it was decided, that the best protocol would be an overnight induction at 25oC with 1mM concentration of IPTG. For the construction of a timeline of the first 6h this time, since the four-hour protocol did not work, a sample from the bacterial culture had been removed every hour, and at the time of sixth hour, the sample taken was processed and separated into soluble and insoluble part. The samples then were analyzed using 8% gel electrophoresis and a western blot technique was performed. For the better visualization of the MW of our protein, we chose to run the gel for extended time (compared to the Figure 23) and as shown in Figure 25, the band is illustrated exactly in the MW we waited.

At the end of the six-hour time point the expression of the soluble SUMO-LbuCas13a was observed. At the same time the quantity of the insoluble SUMO-LbuCas13a has been increased also.

LbuCas13a protein expression procedure with overnight induction, at 25oC using different concentrations of IPTG

After the conformation of the expression conditions, different concentrations of IPTG were tested, in order to: a) determine the most efficient concentration that produces a satisfactory quantity of soluble SUMO-LbuCas13a, b) investigate and understand which concentration offers the best soluble/insoluble ratio. Taking into account the above, five different concentrations of IPTG were used: 1mM, 0.2mM, 0.4mM, 0.6mM and 0.8Mm following the same protocol. Meaning, 1L bacterial cultures induced overnight at 25oC. The final products were analyzed using SDS-PAGE electrophoresis and western blotting methods. The results rendered IPTG concentration among the most important factors for LbuCas13a protein expression. The higher the concentration of IPTG was, the higher the amount of the protein expressed, as depicted in Figures 27, 28. It is also observed that the induction procedure with 1mM IPTG offered the best results.

Purification procedure of SUMO-LbuCas13a protein

The last step for the LbuCas13a production constitutes the purification and digestion of SUMO-LbuCas13a, using the cytiva His trap Ni-NTA column kit and the SUMO protease ULP1, respectively. Both protocols were applied on soluble and insoluble SUMO-LbuCas13a as well. The results were analyzed through SDS-PAGE and western blotting methods. The purification and the cleavage of the SUMO tag were performed successfully on soluble and on inclusion bodies derived protein. The purification and the cleavage of the SUMO tag were performed successfully on soluble and on inclusion bodies derived protein.

Microfluidic device manufacturing

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One of the main objectives of our project was the manufacturing of a flow-focusing microfluidic device. The selected manufacturing process was the 3D-printing. The microfluidic device is demonstrated in Figure 31 and so as the channel geometry.

The 3D printing technique selected was the Fused Deposition Modelling. After printing the whole microfluidic device at once, we observed that the printed object was unable to perform microfluidic device behavior. The second approach with the Fused Deposition Modelling was printing the device into 2 steps. The first step was the printing of all the layers until the last layer of the microfluidic channels. The second step was the printing of the rest layers of the device and finally the construction of the whole device by melting the bottom of the second part and attaching it to the first one. This methodology was implemented also with the Digital Light Projection (DLP) technique for comparison.

For more details Engineering Success (page Cycle 3).

Bulk fluorescence experiments

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Feasibility of LbuCas13a/crRNA system for miRNA detection.

The LbuCas13a/crRNA detection system is based on the LbuCas13 protein which processes crRNA-guided ssRNA recognition that subsequently activates the trans cleavage of nontarget RNA molecules by the LbuCas13a/crRNA machinery. Firstly, the crRNA assembles with the Cas13a and directs it to recognize the target miRNA that are complementary to the spacer region of the crRNA. After target recognition, the Cas13a is activated to cleave nearby RNA nonspecifically such as Reporter FQ5U RNA molecules which are added in the reaction. Specifically, the LbuCas13a digests the fluorophore (FAM)- and quencher (BHQ1)-labeled poly-U reporter probe (FQ5U) getting rid of the fluorescence resonance energy transfer (FRET) effect and releasing the fluorescence signals (Liu et al., 2017).

As described above, the detection system's main components are the LbuCas13a protein, the crRNA specific for the target miRNA and the RNA reporter (FQ5U) necessary for the fluorescence signals.

• According to the SDS-PAGE electrophoresis results (figure), the LbuCas13a demonstrates the predicted MW with an excellent purity of about 95-98%.
• The 3% agarose gel electrophoresis of the in vitro transcribed crRNAs (figure) prove the estimated MW of the in IVT crRNAs as well as their purity.

To efficiently construct a well-characterized LbuCas13a/crRNA based detection system we had to investigate the experimental conditions that affect the reaction performance. The following experiments were conducted to investigate the reaction conditions and provide a strong proof-of-concept of our systems functionality:

• The reaction buffer composition for enabling LbuCas13a collateral activity.
• The RNA reporter (FQ5U) concentration in the reaction, necessary for the fluorescence signal. The results of this experiment are presented at the engineering cycle 2 of the engineering success wiki page (figure 2 of engineering success).
• The time point of fluorescence detection since reaction initiation. The fluorescence capture point should be in the linear part of the curve before the plateau phase for system's best performance. The results of this experiment are presented at the engineering cycle 2 of the engineering success wiki page.
• Standard curve construction which plots the fluorescence intensity of known added miRNA concentrations (standards) in the reaction. This calibration curve can be used for the determination of the target miRNA concentration in unknown samples. The results of this experiment are part of our project's proof-of-concept experiments and thus are presented on the proof-of-concept page of our Wiki.
• Quantification of known added miR-17-3P concentrations in samples utilizing the constructed standard curve. Based on the DIAS platform standard curve (figure) we tested the system's detection accuracy using different reaction samples which contain a specified known concentration of the miR-17-3P. The results of this experiment are part of our project's proof-of-concept experiments and thus are presented on the proof-of-concept page of our Wiki.
• Quantification of known added miR-17-3P concentrations in "non-selective miRNA-enriched complex environments" utilizing the constructed standard curve. The aim of this study is to investigate the LbuCas13a enzyme's ability to efficiently bind with the crRNA and subsequently with the target miRNA in an “artificial environment” which contains a pool of non-specific miRNAs. The results of this experiment are part of our project's proof-of-concept experiments and thus are presented on the proof-of-concept page of our Wiki.
• Relative quantification of miRNA-17-3P in total RNA extracts isolated from 2 different cell lines. The aim of this experiment is to demonstrate the LbuCas13a/crRNA system's feasibility in detecting the miR-17-3P in a pool of total RNAs as well as investigate the target miR-17-3P expression levels in lung cancer cell compared to normal lung cells. The results of this assay are part of our project's proof of concept experiments and thus are presented on the proof-of-concept page of our Wiki.

Reaction buffer composition enabling LbuCas13a collateral activity.

The reaction buffer composition affects the functionality of the LbuCas13a enzyme as well as the stability of the crRNA molecules necessary for the reaction. If the reaction buffer does not contain the necessary components or the appropriate pH, the functionality of the LbuCas13a protein can be minimized reducing the detection system's performance (Shan et al., 2019). According to an extensive literature research 2 reaction buffers were deemed suitable for use (Liu et al., 2017). In addition, we decide to investigate the LbuCas13a activity on the Taq DNA polymerase standard buffer (Taq DNA Polymerase PCR Buffer, Cat. No. 18067-017, invitrogen) to clarify if the LbuCas13a/crRNA reaction can be performed at a standard PCR buffer.

According to our kinetic analysis results, the Buffer B demonstrated inconsistent results which proved that this buffer is not suitable for our LbuCas13a/crRNA reaction. This can be attributed to the low reaction pH (slightly acidic) or to the HEPES buffer composition.

We then conducted comparative experiments between the Buffer A and the Taq DNA Polymerase standard buffer to test the LbuCas13a enzyme's functionality. LbuCas13a (20nM) was incubated with 20nM crRNA and 1μM FQ5U RNA reporter in reaction buffer A or in Taq DNA Polymerase standard buffer respectively. Two different concentrations of a synthetic target miR-17-3p (1nm & 5nM) was added in each reaction tube. The total reaction volumes were 50μl. The mixtures were then incubated in a fluorescence micropate reader (Tekan) at 37 °C for 1 hour with fluorescence measurements (FAM channel, λex 494 nm, λem 518nm) taken every second min. The raw fluorescence intensities generated by the efficient cleavage of the RNA reporter by the LbuCas13a enzyme in each tested reaction buffer are presented on Figures 34 (Reaction Buffer A) and 35 (Taq DNA polymerase standard buffer).

Analyzing the results, we can conclude that the Buffer A provides consistent and reliable results while the common Taq Polymerase standard buffer provides inconsistent results without statistical significance in the observed fluorescence intensities of 1nM and 5nM reaction tubes. In the latter buffer, the fluorescence intensity is not proportionally elevated according to the target miRNA concentration (1nM or 5nM) added in the reaction. These results likely prove that the MgCl2 concentration added in Buffer A is necessary for the proper functionality of the LbuCas13a enzyme. The effect of the Mg2+ ion on the Cas13 processing efficiency has been previously reported on literately for the type VI-D Cas13d ribonuclease (Zhang et al., 2018). In addition, since the pH and the KCl concentrations are the same between the 2 buffers, the differences of Tris HCl buffer concentration could probably affect the system's functionality. Improper reaction buffer could cause a significant background signal implicated the analysis. However, to draw safe conclusions further experiments should be conducted.

Investigation of the RNA reporter concentration required for the detection assay.

Since we concluded that the Buffer A ensures the enzyme's functionality, we had then to evaluate the RNA Reporter concentration needed to achieve the best detection system's performance. Reaction tubes were prepared with 20nM LbuCas13a, 20nM crRNA, 1nM target miR-17-3P and different concentrations of RNA Reporter (0.1nM, 0.25nM, 0.5 nM, 0.75nM & 1nM). The total reaction volumes were 50μl. The mixtures were then incubated in a fluorescence micropate reader (Tekan) at 37 °C for 1 hour with fluorescence measurements (FAM channel, λex 494 nm, λem 518nm) taken every second min as previously. After data acquisition we proceeded to a Michaelis-Menten analysis exploited the in-silico analysis provided on the model page. Further information regarding the Michaelis-Menten analysis of our system is provided on the measurement page.