Difference between revisions of "Part:BBa K3806014"

Revision as of 16:58, 20 October 2021

Theophylline-binding aptazyme regulating lacZ expression (cRBS). With T7 promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3204
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2321
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Sensing small molecules is the foundation of many applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Synthetic genetic switches are a promising tool to detect and quantify small molecules. Conventional synthetic biology-based biosensors to detect small molecules are based on transcription factors. However, transcription factors are not easily reprogrammable with respect to ligand selectivity. On the contrary, antibody-based biosensors can be easily developed into new sensing capabilities. Unfortunately, these types of sensors are not suitable to detect low molecular weight compounds. RNA-based biosensors are an interesting alternative since they present remarkable flexibility to be engineered into sensing a wide range of analytes with high sensitivity and selectivity. In particular, aptazymes, ligand-regulated self-cleaving ribozymes, are of special interest as cleavage of the aptazyme can be coupled to regulated gene expression in vivo or in vitro. Moreover, newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse ligands.

The TU Delft 2021 team provides the iGEM community with a novel genetic switch construct (BBa_K3806014) in which an aptazyme sequence is fused to the lacZ reporter gene for converting ligand concentration to a colorimetric read-out. The construct is designed to be modular and serves as a template to engineer other ligand-specific genetic circuits by swapping the aptazyme domain. It was demonstrated that the designed genetic construct can be expressed in a cell-free system by using a known theophylline binding aptazyme [1].

Aptazyme-regulated gene expression mechanism

The aptazyme-regulated expression of lacZ depends on the accessibility of the ribosomal binding site (RBS) for translation initiation. This design choice was inspired by the study of Klauser & Hartig [2] in which the exposure of the RBS for translation initiation is controlled using small RNA switches. In BBa_K3806014, the RBS of lacZ is also sequestered by an antisense helix. After transcription of the DNA template comprising the aptazyme and fused reporter gene, the RBS is hidden in the stem of the aptazyme. When the aptazyme is stabilized upon binding of the ligand, the RBS remains entirely sequestered by its antisense strand, and translation is mostly prohibited. Cleavage of the aptazyme in the absence of the ligand liberates the RBS (Fig. 1). As a result, the ribosome can bind to the RBS and translate the downstream reporter gene to β-galactosidase protein.

Subsequently, β-galactosidase enables a colorimetric change by converting yellow substrate chlorophenol red-b-D-galactopyranoside (CPRG) to the red product chlorophenol red (CPR).

Fig. 1. 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).

Experimenta results

Cleavage characterization of the aptazyme-regulated genetic construct in T7 transcription reaction

The ligand-dependent cleavage activity of the theophylline aptazyme in a T7 transcription reaction was characterized by RT-PCR. First, the genetic construct was expressed in T7 transcription reaction for 1 hour (Fig. 2, step 1) followed by a reverse transcription (RT) reaction to convert the transcribed mRNA to cDNA (step 2). These cDNA products were then amplified using two primer sets. One that flanks the cleavage site (uncleaved region) and one that flanks a region downstream the cleavage site (control region). In the former case, it should be noted that only the uncleaved mRNA fraction will be PCR amplified (step 3). As a final step, the PCR product was visualized on an agarose gel (step 4).

If the cleavage activity is maintained using the lacZ fused aptazyme, it is expected that a lower band intensity with decreasing concentration of theophylline is seen. This would mean that cleavage occurred and the forward primer flanking the cleavage site was unable to bind to the 5’ of the cDNA to produce a PCR product (Fig. 2). The region downstream the cleavage point was intended to be used as a control for transcription, RT, PCR, and loading in the agarose gel, as band intensity should be similar between samples regardless of theophylline addition.

Fig. 2 Scheme of the 4 steps of the cleavage characterization procedure (Left). Agarose gel analysis of the theophylline-dependent cleavage activity of the aptazyme after T7 transcription. (A) PCR product after amplification of the uncleaved region (the uncleaved fraction is displayed in the bottom of the gel). (B) PCR product after amplification of the control region. Lanes: (Ladder) dsDNA ladder. (1) NC: negative control without DNA template. (2) 0 mM theophylline (3) 1 mM theophylline. (4) 5 mM theophylline.

The resulting agarose gel after the above-mentioned procedure can be seen in Fig. 2. The band intensity, and thus the uncleaved fraction of the aptazyme, is increasing with increasing theophylline concentrations. This means that the aptazyme preserves its theophylline-dependent cleavage activity in the T7 transcription reaction (Fig. 2). Additionally, the fusion of the lacZ gene to the aptazyme sequence does not hinder aptazyme self-cleavage. Based on these results, it is proved that the aptazyme presents a ligand-dependent self-cleavage activity after transcription in a T7 reaction buffer.

Expression of genetic construct in absence and presence of ligand

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.
The genetic construct was expressed in the absence and presence of theophylline in PURExpress using CPRG as substrate. The product formation was determined by measuring the CPR absorbance peak at 575 nm (Fig. 3).

Expression of genetic construct in absence and presence of ligand

Fig 3. Expression of BBa_K3806014 in PURExpress with different ligand concentrations of 0 and 5 mM theophylline. CPR production was quantified as a measure of absorbance at 575 nm.
Decreased absorbance is observed at 5 mM concentration of theophylline compared to 0 mM theophylline. Considering the cleavage characterization in T7 transcription reaction in the previous experiment, it is suspected that the colorimetric read-out, and therefore expression, is ligand-responsive. However, in alternative experiments inhibitory effects of theophylline on expression in PURExpress was observed, leaving the chance that the ligand-dependent read-out in this experiment is a combination of both theophylline inhibition and ligand-responsive aptazyme cleavage.

Ligand-regulated expression on paper

Next, the ligand-regulated expression system was tested on a paper support. Cell-free system reactions were embedded and freeze dried onto paper discs. After 5 days of storing at room temperature, the paper discs were rehydrated with water or 5 mM theophylline, and incubated at 37 °C for 24 hours (Fig. 4). As expected, there was no visual color difference between the paper discs shortly after rehydration (Fig. 4, 0 h). After 24 hours of expression an increased color change is visible for the sample without theophylline, compared to the 5 mM theophylline (Fig. 4, 24 h). This is in correspondence with the results observed in the previous experiment in liquid.

Fig 3. Expression of BBa_K3806014 in PURExpress with different ligand concentrations of 0 and 5 mM theophylline. CPR production was quantified as a measure of absorbance at 575 nm.

References

[1] Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.
[2] Klauser, B., & Hartig, J. S. (2013). An engineered small RNA-mediated genetic switch based on a ribozyme expression platform. Nucleic acids research, 41(10), 5542-5552.

===Functional Parameters=== BBa_K3806014 parameters

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 ===Experimenta results===
-<HTML><h3>Cleavage characterization of the aptazyme-regulated genetic construct in T7 transcription reaction<h3>
+<HTML><h3>Cleavage characterization of the aptazyme-regulated genetic construct in T7 transcription reaction</h3>
 </html>
 The ligand-dependent cleavage activity of the theophylline aptazyme in a T7 transcription reaction was characterized by RT-PCR. First, the genetic construct was expressed in T7 transcription reaction for 1 hour (Fig. 2, step 1) followed by a reverse transcription (RT) reaction to convert the transcribed mRNA to cDNA (step 2). These cDNA products were then amplified using two primer sets. One that flanks the cleavage site (uncleaved region) and one that flanks a region downstream the cleavage site (control region). In the former case, it should be noted that only the uncleaved mRNA fraction will be PCR amplified (step 3).  As a final step, the PCR product was visualized on an agarose gel (step 4).