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Engineering Success

During our project, we encountered various obstacles. By applying the engineering cycle, we were able to improve the optimal reaction conditions of the in vitro evolution of aptazymes, discover a cell-free system compatible with our desired expression system and realize an improvement of our genetic circuit’s expression.

Introduction

With the goal of making hidden hunger visible, we designed a modular, quantitative, and accessible rapid diagnostic test (RDT) for vitamin deficiencies. We engineered vitamin-specific aptazyme biosensors through a novel in vitro evolution method [1]. Thereafter, for a colorimetric read-out of vitamin concentrations, an aptazyme-based genetic circuit was designed to regulate the expression of a reporter gene using a cell-free system on paper support. Throughout the project, we went through several engineering cycles (Design → Build → Test → Learn → Repeat, Fig. 1) for optimizing our RDT. First, we performed an engineering cycle to establish the optimal reaction conditions for the in vitro evolution of aptazymes. In the second engineering cycle, we explored different cell-free expression systems to finally discover the appropriate system for our genetic circuit. The third and last cycle focused on improving the expression of our genetic construct, successfully using the engineering cycle to create and characterize new parts.

Fig. 1 General stages of the engineering cycle.

Engineering cycle 1: adjusting reaction conditions for in vitro evolution of folate biosensors

Folate (vitamin B9) is crucial during early pregnancy to reduce the risk of birth defects of the fetus’ brain and spine. Due to the relevance of this vitamin, we engineered a folate-regulated biosensor through in vitro evolution [1]. Each round of the in vitro evolution process consists of a transcription, reverse transcription, ligation, and PCR steps. During transcription, the cleavage of RNA molecules is regulated by the presence of the ligand, folate in this case. Importantly, the addition of the ligand should not interfere with transcription. Below, we describe the engineering cycle we underwent to set up the reaction conditions using folate as a ligand.

First iteration

Design: folate solubility

Folic acid is only slightly soluble in water, 0.0016 mg/ml (3.6 μM) at 25 °C [2]. Given this low solubility, water could not be used as a solvent to achieve the desired final concentration of 100 µM folate^* in ligand selection rounds. Nonetheless, solubility of folate in 1 M NaOH is significantly higher than in water, 50 mg/ml (113.3 mM) [3]. Therefore, we decided to prepare a 2 mM folate stock solution in 1 M NaOH. To reach a folate concentration of 100 μM in the transcription reaction, 1 μl of this stock solution was used.

Concerned that an increase in pH could have an impact on the transcription reaction, we set up an experiment to test this solvent’s effect. In order to do so, we used a known theophylline aptazyme sequence (here named gB-Theo) as positive control, which was previously discovered by Townshend et al. [1].

^*Reasoning behind the choice of using 100 μM of folate:
The DRIVER process enriches sequences able to bind to the desired ligand. Functional aptazymes represent a small fraction of the initial library. Moreover, it is stated that the improvement in the sensitivity of these sequences improves with further rounds of evolution. Therefore, we decided to use a high concentration of ligand, 100 uM, to enrich sequences with affinity even if such affinity was low.

Build and Test

Transcription reactions of the gB-Theo were performed with and without the addition of 1 μl of 1 M NaOH, followed by a Urea-PAGE gel to visualize the transcription products (Fig. 2).

Fig. 2 Urea-PAGE gel to assess the influence of 1 μl 1M NaOH on transcription. Lanes: (L) denatured dsRNA ladder, (1) Negative control: no DNA template. From lane 2 to lane 5, gB-Theo was used as a DNA template: (2) 1 μl of 1 M NaOH and 0 mM theophylline, (3) 1 μl of 1 M NaOH and 10 mM theophylline, (4) 0 mM theophylline, and (5) 10 mM theophylline. Lane 4 and 5 were used as positive controls.

Learn: high pH inhibits transcription

From this experiment, we realised that the chosen conditions were not appropriate for transcription to occur. Most likely, adding 1 μl of 1 M NaOH to the reaction caused an increase in the pH that was incompatible with transcription (Fig. 2, lanes 2 and 3). To address this issue, we entered the next iteration of this engineering cycle.

Second iteration

Design: compromise between pH and solubility

To avoid transcription inhibition and still use folate at the desired concentration, we tried to find a compromise between folate solubility and the pH of the folate solution. We intended to do this by preparing folate solutions at different concentrations and adjusting pH until folate was completely dissolved. Afterwards, these solutions were designed to be tested in transcription reactions.

Build

In the building phase, we prepared a less concentrated folate stock solution of 0.66 mM. With this concentration, we observed that once we reached a pH of 8, folate was completely dissolved (Fig. 3). The T7 RNA polymerase reaction buffer has a pH of 7.9 [4], suggesting that transcription should no longer be affected when adding the newly prepared solution. Then, to reach a folate concentration of 100 μM in the transcription reaction, we used 3 μl of this stock solution.

Fig. 3 Building phase. Adjusting pH of a 0.66 mM folate solution.

To further test the effect of the folate solution’s pH, if not properly adjusted to 8, we prepared two additional solutions at pH 8.5 and 9.

Test

Again, transcription reactions were set up and a Urea-PAGE gel was run to visualize the transcription products (Fig. 4).

Fig. 4 Urea-PAGE gel to assess the influence of the pH of the folate solutions on transcription. From lane 2 to lane 7, gB-Theo was used as the DNA template. Lanes: (L) ssRNA ladder, (1) Negative control: no DNA template, (2) 1 μl of 1 M NaOH, (3) 3 μl of folate solution at pH 8.5, (4) 3 μl of folate solution at pH 9, (5) 3 μl of folate solution at pH 8, (6) 3 μl of folate solution at pH 8 and 10 mM theophylline, and (7) Positive control: without ligand.

Success!

Our results in Fig. 4 demonstrated that transcription was possible using all the tested folate solutions (lanes 3 to 6) as levels of transcription were comparable to those observed in the positive control, a sample without the addition of folate (lane 7). The reaction conditions were successfully set up and ready to start the in vitro evolution rounds!

Engineering cycle 2: in search for a cell-free expression system for AptaVita

With the goal of developing a robust, safe, modular, and accessible vitamin detection kit, a cell-free system, freeze-dried on paper support, was chosen as the desired expression platform. For more information on the reasoning about our design choice, visit our Design page. In this section, we describe how different cell-free expression systems were tested and how we went through several iterations of the engineering design cycle to identify the suitable expression system for our genetic circuit.

First iteration

Design: wheat germ based cell-free expression system

Firstly, we created a design for expression of lacZ in a eukaryotic wheat germ based cell-free system. In this design, the theophylline aptazyme sequence was added to the 3’ untranslated region, after the stop codon of the lacZ gene and before the start of the barley yellow dwarf virus 3' untranslated region (BYDV 3’UTR), a known translation enhancer sequence [5]. Cleavage of the aptazyme means that the BYDV 3’UTR enhancer is detached from the mRNA, hindering translation, whereas a stabilized aptazyme results in enhancement of translation upon binding of the ligand (Fig. 5).

Fig. 5 Schematic representation of the eukaryotic aptazyme-regulated expression mechanism. The aptazyme is located between the stop codon of the lacZ gene and the start of the BYDV 3’UTR. Upon binding of the ligand, the aptazyme is stabilized preventing self-cleavage. This way, the BYDV 3’UTR enhancer sequence is preserved, resulting in higher levels of expression (top). Cleavage of the aptazyme means that the BYDV 3’UTR enhancer is detached from the mRNA, resulting in lower levels of expression (bottom).

Build and Test

The sequence of the above mentioned eukaryotic genetic construct was synthesized and reactions were assembled according to Protocol: wheat germ of Cell-free expression system protocol. Immediately after the assembly of all reaction components, a color change of the substrate from yellow to purple was observed in all wells, including the negative control (Fig. 6).

Fig. 6 Color change after reaction assembly using a wheat germ based cell-free expression system. Wells: (1) Negative control without DNA template, purple; (2) 0 mM theophylline, purple; (3) 0.5 mM theophylline, purple; and (4) 6 mM theophylline, purple. (1-3) with DNA template. CPRG was used as a substrate to a final concentration of 0.6 mg/ml.

Learn: background activity on CPRG in the wheat germ extract

After testing the system, two reasons led us to think that the observed color change was due to some background activity that degrades CPRG in the wheat germ extract, rather than due to gene expression from our genetic construct:

1. The color change appeared within seconds. This means that there should be something already present in the reaction mixture that catalyzes or degrades the CPRG, as time passed after the assembly was not enough for gene expression to happen.
2. A change of color from yellow to purple was also observed in the negative control, which did not contain the DNA template (Fig. 6). This means that cleavage of CPRG to CPR was not caused by lacZ expression from the DNA template. To confirm this, we added CPRG to the wheat germ extract, and the color change was again observed.

For these reasons, we hypothesized that the cleavage of CPRG was catalyzed by a molecule present in the wheat germ extract. This cell-free expression system is prepared from a wheat germ extract, implying that not only the components required for transcription and translation are present, but also other components, such as enzymes that can potentially degrade CPRG. Altogether, we learned that this system is not compatible with our envisioned application using CPRG.

Second iteration

Design: PUREfrex2.0 cell-free expression system

In order to avoid undesired side reactions, we switched to a PURE (Protein synthesis Using Recombinant Elements) based cell-free system. As opposed to cell extracts, PURE systems are composed of a defined number of purified and recombinant components needed for in vitro transcription and translation [6]. Therefore, we expect less unintended conversion of CPRG due to background reactions.

In particular, we used the PUREfrex2.0 system (GeneFrontier). This system contains purified transcription and translation components from E. coli [6]. Consequently, we had to design a new genetic construct for lacZ expression in a prokaryotic system (BBa_K3806014). In this design, the reporter gene expression depends on accessibility of the ribosomal binding site (RBS) for translation initiation. Cleavage of the aptazyme in the absence of the ligand frees the RBS resulting in translation of the downstream reporter gene. On the other hand, when the ligand is present there is no cleavage, keeping the RBS hidden in the stem of the aptazyme, repressing translation (Fig. 7). In essence, the more ligand we add, the less color change we would expect. For more details about this design, visit our Design page.

Fig. 7 Aptazyme-regulated gene expression mechanism. Binding of the ligand renders a catalytically inactive aptazyme, the RBS remains entirely sequestered by its antisense strand, repressing translation (left). In the absence of the ligand, self-cleavage of the aptazyme frees the RBS, resulting in the binding of the small ribosomal subunit and initiation of translation (right).

Build and Test

The genetic construct was synthesized (and sequenced verified by Twist Bioscience) and reactions were assembled according to Protocol: PUREfrex® 2.0 of the Cell-free expression system protocol (using CPRG as substrate and on paper support). The results shown in Fig. 8 were obtained after incubation at 37 °C for 3 hours.

Fig. 8 Paper-based reactions using PUREfrex2.0 following a 3 hours incubation at 37 °C. Wells: (top left) without DNA template and 0 mM theophylline, red; (top right) without DNA template and 7 mM theophylline, yellow; (bottom left) with DNA template and 0 mM theophylline, red; and (bottom right) with DNA template and 7 mM theophylline, yellow. The DNA template was added at a final concentration of 5 nM. CPRG was used as a substrate to a final concentration of 0.6 mg/ml.

Learn: cleavage of CPRG in PUREfrex2.0

As mentioned in the design section of this iteration, this system is designed such that the more ligand added, the less color change is expected. In the presence of DNA, this behavior was observed (Fig. 8, bottom). However, the same behavior was seen in the absence of DNA template (Fig. 8, top), meaning that the color change was not a result of lacZ expression from our genetic construct. In the absence of DNA and the presence of theophylline no color change was observed.

Two hypotheses were derived from these observations and tested experimentally:

1. The negative controls are contaminated with DNA. However, this hypothesis was ruled out as experiments were repeated with new reagents, and the result remained the same.
2. CPRG can spontaneously be converted into CPR, a process that would be inhibited by theophylline. This hypothesis was discarded as a serial dilution of CPRG in water was prepared and no color change was observed.

With all the possible combinations being tested, we hypothesized that there should be a component in PUREfrex2.0 that cleaves CPRG into CPR; this conversion is however inhibited in the presence of theophylline. In conclusion, CPRG is not compatible with our PUREfrex2.0 expression system set-up.

Third iteration

Design: X-gal as substrate

Besides CPRG, β-galactosidase is able to convert other substrates, such as X-gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside). β-galactosidase X-gal, releasing the substituted indole that spontaneously dimerizes to give an insoluble, blue product [7]. We decided to use X-gal as the reaction substrate with the hope that no side reactions would take place.

Build and Test

Reactions were assembled according to Protocol: PUREfrex® 2.0 of Cel-free expression system protocols. X-gal, dissolved solely in DMSO, was used as a substrate. As DMSO could potentially interfere with the aptazyme function [8], it was added 2 hours after the beginning of lacZ expression. The image shown in Fig. 9, was taken after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight.

Fig. 9 Image of PUREfrex2.0 reactions using X-gal as substrate. Samples: (1) without DNA template and 0 mM theophylline, transparent; (2) without DNA template and 7 mM theophylline, transparent; (3) with DNA template and 0 mM theophylline, blue; and (4) with DNA template and 7 mM theophylline. The DNA template was added at a final concentration of 5 nM. X-gal was used as a substrate to a final concentration of 8.3 mg/ml.

Learn: X-gal can be used as substrate in combination with PUREfrex2.0

Under the conditions tested, no color change could be observed in the negative control (no DNA added) in the presence or absence of the ligand (Fig. 9). These results suggested that X-gal can be utilized as a substrate when using PUREfrex2.0 as the expression system.

Success! But…

Despite the positive results, X-gal dissolved in DMSO can only be added after expression to prevent interference with the aptazyme. This presents disadvantages as longer incubation times are required and the kinetic trace of expression can not be monitored. Moreover, CPRG is reported to be a more sensitive substrate than X-gal [9]. Considering that having both a rapid and quantitative test is an essential requirement of our design, we dove into the fourth and last iteration of this engineering cycle.

Fourth iteration

Design: PURExpress cell-free expression system

After performing literature research on the use of CPRG in combination with cell-free systems and theophylline, we found a recently published article in which a theophylline biosensor was built using CPRG for a colorimetric readout using PURExpress (New England Biolabs) [10]. Considering that no side reactions happened in this study, we decided to try out the PURExpress as an alternative option to our setup when using CPRG as a substrate.

Build and Test

Reactions were assembled according to Protocol: PURExpress of Cel-free expression system protocols. The image shown in Fig. 10, was taken after incubation at 37 °C for 1 hour.

Fig. 10 Image of PURExpress reactions using CPRG as substrate following 1 hour incubation. Samples: (1) without DNA template and 0 mM theophylline, yellow; (2) without DNA template and 6 mM theophylline, yellow; (3) with DNA template and 0 mM theophylline, red; and (4) with DNA template and 6 mM theophylline, orange. The DNA template was added at a final concentration of 5 nM. CPRG was used as a substrate to a final concentration of 0.6 mg/ml. In this reaction, 6 mM of theophylline was used (7 mM in Fig. 9) to be able to adjust to the final working volume.

Success!

As can be seen in Fig. 10, no color change could be observed in the negative controls, suggesting that CPRG can be utilized as a substrate when using PURExpress as the expression system. We finally found the appropriate cell-free expression system for our desired substrate!

Engineering cycle 3: improving expression of the genetic construct

First iteration

Design, build, and test: comparing expression levels of the aptazyme-regulated construct to a control

To get a better understanding of our system, we compared the expression levels of our aptazyme-regulated construct (cRBS, BBa_K3806014) to a control part without an aptazyme switch. We used the BBa_I732017 part from the iGEM repository, that contains a lacZ gene and we fused a T7 promoter to enable expression. We performed the experiment in the absence of theophylline, which is expected to give the highest output signal of the system. It should be noted that this assay was performed using PUREfrex2.0 and X-gal because of limited resources in PURExpress. Nevertheless, for the purpose of this experiment, both systems can be used as only the end-point measurement is needed. The image shown in Fig. 11A, was taken after expression at 37 °C for 2 hours, and incubation with X-gal at room temperature overnight. In addition, the amount of X-gal produced was measured via absorbance at 600 nm (Fig. 11B).

Fig. 11 Comparison of X-gal production levels using the BBa_I732017 (iGEM repository) and cRBS constructs. (A) Image of the PUREfrex2.0 reaction after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight. The DNA templates were added at a final concentration of 1 nM. (B) Quantification of X-gal production as measured by absorbance at 600 nm. Data represents the mean ± SE (n = 3). The asterisk indicates a p-value ˂ 0.05 in Student's t-test.

Learn: low expression levels

The absorbance values of the cRBS construct indicated minor expression after 2 hours (Fig. 11, cRBS). Compared to control construct (Fig. 11, BBa_I732017), expression was 4.4 times lower. This means that the dynamic range of our biosensor was limited to this maximum expression level, while having a higher dynamic range would help to distinguish smaller differences in the ligand concentration. Based on this observation, we hypothesized that even if the aptazyme was cleaving in the absence of the ligand, the cleaved fragment could remain attached to the RBS, prohibiting translation (Fig. 12). This hypothesis led us to a new design.

Fig. 12 Schematic representation of the hypothesized scenarios after aptazyme self-cleavage. In the first case, the antisense fragment detaches upon self-cleavage of the aptazyme, liberating the RBS. In the second case, the antisense fragment stays attached to the complementary sequence of the aptazyme including the RBS. Therefore translation is hindered even when the aptazyme is cleaved.

Second iteration

Design: new genetic construct

In our effort to improve the expression levels of our genetic construct, we designed a new construct in which the number of complementary bases in the antisense strand fragment are reduced to 9 bp (11 bp in our previous design), such that only four nucleotides of the RBS are sequestered (Fig. 13). By lowering the number of complementary base pairs, we expected that the detachment of the antisense strand from the RBS occurs more frequently, increasing the expression of lacZ in the absence of the ligand. We created the new design by performing a PCR with a forward primer that changes the C and T (U in the aptazyme) nucleotides of the antisense strand into G and A, respectively, resulting in a partial mismatch to the RBS. Find the primers in the Cell-free expression system notebook on the 28th September, 2021.

Fig. 13 Secondary structure of the two antisense-aptazyme-RBS designs. (A) Eleven nucleotides are complementary to the antisense strand, covering the complete RBS. (B) Nine nucleotides are complementary to the antisense strand, covering four nucleotides of the RBS. The RBS is shaded in magenta and the two modified nucleotides of the antisense strand are pointed with a balck arrow.

Build and Test

We compared the lacZ expression levels between the old (cRBS, BBa_K3806014) and the new (semi-cRBS, BBa_K3806016) designs in the absence and presence of theophylline. To have a more accurate analysis that could not be done with X-gal, PURExpress and CPRG were used as the expression system and substrate, respectively. The product formation was determined by measuring the absorbance at 575 nm over time (Fig. 14).

Fig. 14 Colorimetric response comparison between the cRBS and semi-cRBS designs. CPR production was quantified as a measure of absorbance at 575 nm. (A) During 4 hours of expression. The area enclosed between the absorbance curves corresponding to 0 and 5 mM theophylline is shaded in pink and green for the cRBS and semi-cRBS, respectively. (B) During the first 1.5 hours of expression. The decrease in expression due to theophylline addition is indicated with an arrow.

For both constructs, the area enclosed between the absorbance curves corresponding to 0 and 5 mM theophylline was calculated (Fig. 14A). This is an indicator of both (i) the dynamic range of the biosensor: absorbance difference between the smallest and highest ligand concentration (A0mM - A5mM), and (ii) the response time after 4 hours of expression. Units = absorbance units (Au) x h. In addition, we determined the decrease in the expression level caused by the addition of 5 mM theophylline after 1.5 hours of expression (Fig. 14B):

Success!

For the semi-cRBS, the calculated area was 1.03 h x Au, which compared to the 0.40 h x Au displayed by the cRBS design, results in a 2.6-fold increase. Likewise, when using the semi-cRBS construct, the addition of 5 mM theophylline caused a 33.3% decrease in absorbance already after 1.5 hours of expression compared to the 6.1% decrease in the cRBS design. This indicates a larger dynamic range and shorter response time of the biosensor when using the semi-cRBS construct. Hence, we successfully engineered our system for a faster and more quantitative read-out!

References

1. Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. & Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.
2. National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 135398658, Folic acid. Retrieved October 3, 2021 from https://pubchem.ncbi.nlm.nih.gov/compound/Folic-acid
3. Sigma Aldrich. F7876 Folic acid specification sheet. Retrieved October 3, 2021 from https://www.sigmaaldrich.com/NL/en/product/sigma/f7876?cm_sp=Insite-_-caSrpResults_srpRecs_srpModel_folic%20acid-_-srpRecs3-1
4. New England Biolabs. T7 RNA Polymerase. Retrieved October 3, 2021 from https://www.bioke.com/webshop/neb/m0251.html
5. Wang, S., Browning, K. S., & Miller, W. A. (1997). A viral sequence in the 3′‐untranslated region mimics a 5′ cap in facilitating translation of uncapped mRNA. The EMBO journal, 16(13), 4107-4116.
6. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature biotechnology, 19(8), 751-755.
7. Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). lacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein science : a publication of the Protein Society, 21(12), 1792–1807.
8. Townshend, B. (2021). CleaveSeq: Scalable characterization of ribozyme-based RNA biosensors. Protocol (Version 1) available at Protocol Exchange [https://doi.org/10.21203/rs.3.pex-1346/v1]
9. Möckli, N., & Auerbach D. (2004). Quantitative beta-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system. Biotechniques, 36(5), 872-6.
10. Nguyen, P.Q., Soenksen, L.R., Donghia, N.M. et al. (2021).Wearable materials with embedded synthetic biology sensors for biomolecule detection.Nature biotechnology, 1.

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