iGEM 2021 — Team TUDelft

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# Team:TUDelft/Notebook

AptaVita

AptaVita

[![homelogo](https://static.igem.org/mediawiki/2021/c/c9/T--TUDelft--navbar_full%2BTU.png "homelogo")](https://2021.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2021.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2021.igem.org/Team%3ATUDelft/Collaborations)
[Attributions](https://2021.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2021.igem.org/Team%3ATUDelft/Sponsors)

Project

[Project Description](https://2021.igem.org/Team%3ATUDelft/Description)
[Design](https://2021.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2021.igem.org/Team%3ATUDelft/Engineering)
[Results](https://2021.igem.org/Team%3ATUDelft/Results)
[Hardware](https://2021.igem.org/Team%3ATUDelft/Hardware)
[Contribution](https://2021.igem.org/Team%3ATUDelft/Contribution)
[Sequencing analysis](https://2021.igem.org/Team%3ATUDelft/Software)
[Partnership](https://2021.igem.org/Team%3ATUDelft/Partnership)
[Sustainable Development](https://2021.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Protocols](https://2021.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2021.igem.org/Team%3ATUDelft/Notebook)
[Parts](https://2021.igem.org/Team%3ATUDelft/Parts)

Model

[Model](https://2021.igem.org/Team%3ATUDelft/Model)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Public Engagement](https://2021.igem.org/Team%3ATUDelft/Communication)
[Proposed Implementation](https://2021.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2021.igem.org/Team%3ATUDelft/Safety)
[Entrepreneurship](https://2021.igem.org/Team%3ATUDelft/Entrepreneurship)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3806)
[Medal Criteria](https://2021.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2021.igem.org/Team%3ATUDelft/Awards)

# Lab Notebook

This page provides notebooks of all wet lab modules where we report on our experiments throughout the 2021 iGEM competition.

* [DRIVER](#DRIVER)
* [CFS](#CFS)
* [EMSA](#Partnership)
* [Microfluidics](#Microfluidics)

Notebook 1 - *In vitro* evolution of aptazymes using DRIVER

If the pdf does not show, click [here](https://static.igem.org/mediawiki/2021/6/6c/T--TUDelft--DRIVER_Notebook_DRIVER.pdf).

To see the DRIVER rounds’ spreadsheet mentioned in the notebook, click [here](https://static.igem.org/mediawiki/2021/1/11/T--TUDelft--DRIVER_Notebook_Rounds_Spreadsheet.pdf).

Notebook 2 - Aptazyme-regulated reporter gene expression using cell-free systems

If the pdf does not show, click [here](https://static.igem.org/mediawiki/2021/4/48/T--TUDelft--CFS_notebook.pdf).

Notebook 3 - Electrophoretic Mobility Shift Assay

If the pdf does not show, click [here](https://static.igem.org/mediawiki/2021/7/7c/T--TUDelft--Partnership_notebook.pdf).

Notebook 4 - Microfluidic chip printing

If the pdf does not show, click [here](https://static.igem.org/mediawiki/2021/4/4a/T--TUDelft--Microfluidics_Notebook.pdf).

## A big thank you to our sponsors!

[![TU Delft](https://static.igem.org/mediawiki/2021/8/85/T--TUDelft--TUDelft_Logo.png "TU Delft")](http://tudelft.nl/en)
[![](https://static.igem.org/mediawiki/2021/f/ff/T--TUDelft--BT_Logo.png "Biotechnology")](https://www.tudelft.nl/en/faculty-of-applied-sciences/about-faculty/departments/biotechnology/)
[![TU Delft Bionanoscience Department](https://static.igem.org/mediawiki/2021/5/53/T--TUDelft--BN_Logo.png "Bionanoscience")](https://www.tudelft.nl/en/faculty-of-applied-sciences/about-faculty/departments/bionanoscience/)
[![Faculty of Applied Sciences](https://static.igem.org/mediawiki/2021/f/f4/T--TUDelft--AS_Logo.png "Faculty of Applied Sciences")](https://www.tudelft.nl/en/faculty-of-applied-sciences/)

[![Genefrontier](https://static.igem.org/mediawiki/2021/1/10/T--TUDelft--Genefrontier-highresolution_Logo.png "Genefrontier")](https://www.genefrontier.com/)
[![TU Delft Bioengineering Institute](https://static.igem.org/mediawiki/2021/9/98/T--TUDelft--BioEng_Logo.png "TU Delft Bioengineering Institute")](https://www.tudelft.nl/bioengineering/)
[![Delft Health Initiative](https://static.igem.org/mediawiki/2021/f/fa/T--TUDelft--DelftHealthInitiative_Logo.png "Delft Health Initiative")](https://www.tudelft.nl/health)
[![BASF](https://static.igem.org/mediawiki/2021/e/e8/T--TUDelft--BASF_Logo.png "BASF")](https://www.basf.com)

[![Simonis](https://static.igem.org/mediawiki/2021/c/c3/T--TUDelft--Erasmus_Logo.png "Erasmus MC")](https://www.erasmusmc.nl/)

[![SkylineDx](https://static.igem.org/mediawiki/2021/b/bc/T--TUDelft--SkylineDx_Logo.png "SkylineDx")](https://www.skylinedx.com/home)
[![V.O. Patents & Trademarks](https://static.igem.org/mediawiki/2021/9/9f/T--TUDelft--VO_Logo.png "V.O. Patents & Trademarks")](https://www.vo.eu/nl/)
[![Merck](https://static.igem.org/mediawiki/2021/e/ee/T--TUDelft--Merck_Logo.png "Merck")](https://www.merckmillipore.com/NL/en)
[![United Consumers](https://static.igem.org/mediawiki/2021/f/fe/T--TUDelft--UnitedConsumers_Logo.png "United Consumers")](https://www.unitedconsumers.com/energie/)
[![Eurofins](https://static.igem.org/mediawiki/2021/c/ce/T--TUDelft--Eurofins_Logo.png "Eurofins")](https://www.eurofins.nl/nl/)
[![Promega](https://static.igem.org/mediawiki/2021/f/f5/T--TUDelft--Promega_Logo.png "Promega")](https://nld.promega.com)
[![DSM](https://static.igem.org/mediawiki/2021/2/29/T--TUDelft--DSM_Logo.png "DSM")](https://www.dsm.com/nederland/nl_NL/home.html)
[![Medical Delta](https://static.igem.org/mediawiki/2021/f/f2/T--TUDelft--Medical-Delta_Logo.jpg "Medical Delta")](https://www.medicaldelta.nl)

[![SnapGene](https://static.igem.org/mediawiki/2021/7/72/T--TUDelft--Snapgene_Logo.png "Snapgene")](http://www.snapgene.com)
[![Biorender](https://static.igem.org/mediawiki/2021/2/22/T--TUDelft--biorender_Logo.png "Biorender")](https://biorender.com/)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:TUDelft/Engineering

AptaVita

AptaVita

[![homelogo](https://static.igem.org/mediawiki/2021/c/c9/T--TUDelft--navbar_full%2BTU.png "homelogo")](https://2021.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2021.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2021.igem.org/Team%3ATUDelft/Collaborations)
[Attributions](https://2021.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2021.igem.org/Team%3ATUDelft/Sponsors)

Project

[Project Description](https://2021.igem.org/Team%3ATUDelft/Description)
[Design](https://2021.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2021.igem.org/Team%3ATUDelft/Engineering)
[Results](https://2021.igem.org/Team%3ATUDelft/Results)
[Hardware](https://2021.igem.org/Team%3ATUDelft/Hardware)
[Contribution](https://2021.igem.org/Team%3ATUDelft/Contribution)
[Sequencing analysis](https://2021.igem.org/Team%3ATUDelft/Software)
[Partnership](https://2021.igem.org/Team%3ATUDelft/Partnership)
[Sustainable Development](https://2021.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Protocols](https://2021.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2021.igem.org/Team%3ATUDelft/Notebook)
[Parts](https://2021.igem.org/Team%3ATUDelft/Parts)

Model

[Model](https://2021.igem.org/Team%3ATUDelft/Model)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Public Engagement](https://2021.igem.org/Team%3ATUDelft/Communication)
[Proposed Implementation](https://2021.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2021.igem.org/Team%3ATUDelft/Safety)
[Entrepreneurship](https://2021.igem.org/Team%3ATUDelft/Entrepreneurship)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3806)
[Medal Criteria](https://2021.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2021.igem.org/Team%3ATUDelft/Awards)

# 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](#Intro)
* [Engineering cycle 1](#cycle1)
* [Engineering cycle 2](#cycle2)
* [Engineering cycle 3](#cycle3)
* [References](#references)

## 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]](#references). 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.

![Figure of General stages of the engineering cycle](https://static.igem.org/mediawiki/2021/1/11/T--TUDelft--Engineering-cycle.jpg "General stages of the engineering cycle")

*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](https://2021.igem.org/Team%3ATUDelft/Design) [[1]](#references). 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]](#references). 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]](#references). 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]](#references).

\*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).

![Figure of Urea-PAGE gel to assess the influence of 1 μl 1M NaOH on transcription.](https://static.igem.org/mediawiki/2021/7/79/T--TUDelft--Folate-N.jpg "Urea-PAGE gel to assess the influence of 1 μl 1M NaOH on transcription.")

*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]](#references), 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.

![Figure of Building phase. Adjusting pH of a 0.66 mM folate solution.](https://static.igem.org/mediawiki/2021/2/2c/T--TUDelft--pHmeter.jpeg "Building phase. Adjusting pH of a 0.66 mM folate 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).

![Figure of Urea-PAGE gel to assess the influence of the pH of the folate solutions on transcription.](https://static.igem.org/mediawiki/2021/0/06/T--TUDelft--Folate-pH.png "Urea-PAGE gel to assess the influence of the pH of the folate solutions on transcription.")

*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](https://2021.igem.org/Team%3ATUDelft/Design#Freezedry) 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]](#references). 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).

![Figure of  Schematic representation of the eukaryotic aptazyme-regulated expression mechanism.](https://static.igem.org/mediawiki/2021/9/93/T--TUDelft--mechanism-Euk.jpg " Schematic representation of the eukaryotic aptazyme-regulated expression mechanism.")

*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](https://2021.igem.org/Team%3ATUDelft/Experiments). 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).

![Figure of  Color change after reaction assembly using a wheat germ based cell-free expression system.](https://static.igem.org/mediawiki/2021/b/b9/T--TUDelft--Wheatgerm.png "Color change after reaction assembly using a wheat germ based cell-free expression system.")

*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]](#references). 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]](#references). Consequently, we had to design a new genetic construct for *lacZ* expression in a prokaryotic system ([BBa\_K3806014](http://parts.igem.org/Part%3ABBa_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](https://2021.igem.org/Team%3ATUDelft/Design) page.

![Figure of  Aptazyme-regulated gene expression mechanism.](https://static.igem.org/mediawiki/2021/1/11/T--TUDelft--Prokaryotic_mechanism.png "Aptazyme-regulated gene expression mechanism.")

*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](https://2021.igem.org/Team%3ATUDelft/Experiments) (using CPRG as substrate and on paper support). The results shown in Fig. 8 were obtained after incubation at 37 °C for 3 hours.

![Figure of  Paper-based reactions using PUREfrex2.0 following a 3 hours incubation at 37 °C.](https://static.igem.org/mediawiki/2021/a/a8/T--TUDelft--PUREfrexCPRG.png "Paper-based reactions using PUREfrex2.0 following a 3 hours incubation at 37 °C.")

*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]](#references). 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](https://2021.igem.org/Team%3ATUDelft/Experiments). X-gal, dissolved solely in DMSO, was used as a substrate. As DMSO could potentially interfere with the aptazyme function [[8]](#references), 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.

![Figure of  Image of PUREfrex2.0 reactions using X-gal as substrate.](https://static.igem.org/mediawiki/2021/a/ac/T--TUDelft--PUREfrexXgal.png "Image of PUREfrex2.0 reactions using X-gal as substrate.")

*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]](#references). Considering that having both a [rapid and quantitative test](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP) 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]](#references). 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](https://2021.igem.org/Team%3ATUDelft/Experiments). The image shown in Fig. 10, was taken after incubation at 37 °C for 1 hour.

![Figure of   Image of PURExpress reactions using CPRG as substrate following 1 hour incubation.](https://static.igem.org/mediawiki/2021/0/0a/T--TUDelft--PURExpressCPRG.png "Image of PURExpress reactions using CPRG as substrate following 1 hour incubation.")

*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](http://parts.igem.org/Part%3ABBa_K3806014)) to a control part without an aptazyme switch. We used the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_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).

![Figure of   Comparison of X-gal production levels using the BBa_I732017 (iGEM repository) and cRBS constructs.](https://static.igem.org/mediawiki/2021/7/72/T--TUDelft--Part-registry-1.png "Comparison of X-gal production levels using the BBa_I732017 (iGEM repository) and cRBS constructs.")

*Fig. 11 Comparison of X-gal production levels using the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_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](http://parts.igem.org/Part%3ABBa_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.

![Figure of   Schematic representation of the hypothesized scenarios after aptazyme self-cleavage.](https://static.igem.org/mediawiki/2021/a/a3/T--TUDelft--Cases.jpg "Schematic representation of the hypothesized scenarios after aptazyme self-cleavage.")

*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](https://2021.igem.org/Team%3ATUDelft/Notebook).

![Figure of   Secondary structure of the two antisense-aptazyme-RBS designs.](https://static.igem.org/mediawiki/2021/a/a3/T--TUDelft--semi-cRBS-vs-cRBS.jpg "Secondary structure of the two antisense-aptazyme-RBS designs.")

*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](http://parts.igem.org/Part%3ABBa_K3806014)) and the new (semi-cRBS, [BBa\_K3806016](http://parts.igem.org/Part%3ABBa_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).

![Figure of  Colorimetric response comparison between the cRBS and semi-cRBS designs.](https://static.igem.org/mediawiki/2021/6/6a/T--TUDelft--cRBS-semi-cRBS-abs.png "Colorimetric response comparison between the cRBS and semi-cRBS designs.")

*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):

![Figure of  Equation of relative change.](https://static.igem.org/mediawiki/2021/5/50/T--TUDelft--Relative-change.png "Equation of relative change.")

#### 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](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]](https://doi.org/10.21203/rs.3.pex-1346/v1%20)
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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# Team:TUDelft/Experiments

AptaVita

AptaVita

[![homelogo](https://static.igem.org/mediawiki/2021/c/c9/T--TUDelft--navbar_full%2BTU.png "homelogo")](https://2021.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2021.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2021.igem.org/Team%3ATUDelft/Collaborations)
[Attributions](https://2021.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2021.igem.org/Team%3ATUDelft/Sponsors)

Project

[Project Description](https://2021.igem.org/Team%3ATUDelft/Description)
[Design](https://2021.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2021.igem.org/Team%3ATUDelft/Engineering)
[Results](https://2021.igem.org/Team%3ATUDelft/Results)
[Hardware](https://2021.igem.org/Team%3ATUDelft/Hardware)
[Contribution](https://2021.igem.org/Team%3ATUDelft/Contribution)
[Sequencing analysis](https://2021.igem.org/Team%3ATUDelft/Software)
[Partnership](https://2021.igem.org/Team%3ATUDelft/Partnership)
[Sustainable Development](https://2021.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Protocols](https://2021.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2021.igem.org/Team%3ATUDelft/Notebook)
[Parts](https://2021.igem.org/Team%3ATUDelft/Parts)

Model

[Model](https://2021.igem.org/Team%3ATUDelft/Model)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Public Engagement](https://2021.igem.org/Team%3ATUDelft/Communication)
[Proposed Implementation](https://2021.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2021.igem.org/Team%3ATUDelft/Safety)
[Entrepreneurship](https://2021.igem.org/Team%3ATUDelft/Entrepreneurship)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3806)
[Medal Criteria](https://2021.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2021.igem.org/Team%3ATUDelft/Awards)

# Protocols

This section provides a step by step description of the protocols that we used for our experiments. The protocols are categorized per wetlab module.

* [General](#General)
* [DRIVER](#DRIVER)
* [CFS](#CFS)
* [Hardware](#Hardware)
* [EMSA](#Partnership)
* [Microfluidics](#Microfluidics)

General protocols

## Agar plates

1. Dissolve 14.24 g of Lysogeny Broth (LB) agar in 400 ml distilled water.
2. Autoclave the LB agar solution.
3. Cool down the solution before adding antibiotics. To speed up the process, you can hold the bottle under cold tap water while stirring.
4. Supply with the appropriate antibiotics. When using 1000X antibiotic stock solutions, add 1 µl of the antibiotic stock solution per ml of LB agar.
5. Under sterile conditions, pour the LB agar in sterile Petri dishes.
6. Leave the Petri dishes near the flame with the lid partially off for approximately 5 minutes so the agar can solidify.
7. Store at 4 °C until use.

## Antibiotic stock solution

For 1000X antibiotic stock solutions:

1. Weight X grams of the antibiotic of interest.
2. Dissolve in 95% ethanol (Chloramphenicol) or Milli-Q water (other antibiotics).
3. Filter the solution using a 45 µm filter.

## DNA concentration determination

### Using NanoDrop UV-Vis Spectophotometer

1. Press the button *dsDNA* to measure the concentration of double stranded DNA in your samples.
2. Clean the measurement surface with kimwipes and Milli-Q water.
3. Use 1 µl of the same buffer the sample is dissolved in to set the blank.
4. Use 1 µl of sample to measure its concentration. If you have multiple samples, clean the measurement surface in between measurements with kimwipes.

### Using Qubit® dsDNA HS Assay Kits

1. Set up the required number of 0.5 ml tubes for standards and samples. The Qubit® dsDNA HS Assay requires 2 standards. Use only thin-wall, clear, 0.5 ml PCR tubes.
2. Label the tube lids. Note: Do not label the side of the tube as this could interfere with the sample read.
3. Prepare the Qubit® working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. Use a clean plastic tube each time you prepare a Qubit® working solution. Do not mix the working solution in a glass container.
   Note: The final volume in each tube must be 200 µl. Each standard tube requires 190 µl of Qubit® working solution, and each sample tube requires anywhere from 180-199 µl. Prepare sufficient Qubit® working solution to accommodate all standards and samples. For example, for 8 samples, prepare enough working solution for the samples and 2 standards: ~ 200 µl per tube in 10 tubes yields 2 ml of working solution (10 µl of Qubit® reagent plus 1990 µl of Qubit® buffer).
4. Add 190 µl of Qubit® working solution to each of the tubes used for standards.
5. Add 10 µl of each Qubit® standard to the appropriate tube, then mix by vortexing 2–3 seconds. Be careful not to create bubbles. Note: Careful pipetting is critical to ensure that exactly 10 µl of each Qubit® standard is added to 190 µl of Qubit® working solution.
6. Add Qubit® working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 µl. Note: Your sample can be anywhere from 1-20 µl. Add a corresponding volume of Qubit® working solution to each assay tube: anywhere from 180-199 µl.
7. Add each sample to the assay tubes containing the correct volume of Qubit® working solution, then mix by vortexing 2-3 seconds. The final volume in each tube should be 200 µl.
8. Allow all tubes to incubate at room temperature for 2 minutes.
9. Read standards and samples following the procedure appropriate for your instrument.

Protocol adapted from Molecular probes Life Technologies (Qubit® dsDNA HS Assay Kits).

## DNA and RNA electrophoresis

1. Prepare TBE 1X buffer. Take the 10X concentrated TBE and dilute it 10 times with Milli-Q water. For 500 ml, add 50 ml TBE 10X to 450 ml distilled water.
2. Weigh agarose for a 1% gel. For 50 ml, 0.5 g of agarose are necessary.
3. Mix 50 ml TBE 1X buffer with the 0.5 g agarose and heat the solution in a microwave at medium-low power until it is completely dissolved.
4. Wait for the mixture to cool down until it reaches a handable temperature and then add SYBR Safe stain. For a 50 ml gel, add 2 µl of SYBR Safe.
5. Pour the solution into the casting mold with the comb already mounted. Make sure there are no bubbles. Let it solidify (~ 20 minutes).
6. Transfer the gel to the electrophoresis cell, minding the arrow that indicates the direction of DNA migration. Remove the combs and cover it with TBE buffer.
7. Prepare the samples. For DNA samples, add the required volume of 6X DNA loading buffer to the sample (1 µl of Loading Buffer per 5 µl of sample). For RNA samples, add the required volume of 2X RNA loading buffer to the sample (1 µl of Loading Buffer per 1 µl of sample), and incubate the samples at 70 °C for 10 minutes.
8. Load the molecular ladder in the first well (verify the appropriate volume for each marker). Then, load the samples in the remaining wells.
9. Connect the cables following the color code and run at 90 V for 45 minutes or as required.

## Gel & PCR Cleanup (Promega Wizard kit)

### Gel Slice and PCR product preparation

### A. Dissolving the Gel Slice

1. Following electrophoresis, excise the DNA band from the gel and place the gel slice in a 1.5 ml microcentrifuge tube.
2. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65 °C until the gel slice is completely dissolved.

### B. Processing PCR products

1. Add equal volume of Membrane Binding Solution to the PCR product.

### Binding, Washing, and Elution

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 x g for 1 minute. Discard flow-through and reinsert Minicolumn into Collection Tube.
4. Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 x g
   for 1 minute. Discard flow-through and reinsert Minicolumn into Collection Tube.
5. Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 x g for 5 minutes.
6. Empty the Collection Tube and centrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
7. Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
8. Add 30 μl Milli-Q water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 x g for 1 minute.
9. Discard Minicolumn. Measure DNA concentration and store at -20 °C.

Protocol adapted from Promega (Wizard® SV Gel and PCR Clean-Up System).

## Glycerol stocks

1. Prepare a 80% (v/v) glycerol solution and sterilize by autoclaving.
2. Inoculate 10 ml LB liquid bacterial culture containing the appropriate antibiotic. Incubate at 37 °C overnight, shaking at 250 rpm.
3. Add 250 µl of the 80% glycerol solution to a sterile cryotube.
4. Add 750 µl of the liquid bacterial culture to the tube and mix well.
5. Store at -80 °C.

## Lysogeny Broth liquid medium

1. Dissolve Lysogeny Broth (LB) powder in distilled water according to manufacturer instructions.
2. Autoclave the LB solution.
3. Store at room temperature until use.

## Liquid bacterial culture inoculation

1. Prepare as many 50 ml falcon tubes as needed. Do not forget to prepare a negative control.
2. In sterile conditions, add 10 ml of LB liquid medium to each falcon tube.
3. Supply with the appropriate antibiotics. When using 1000X antibiotic stock solutions, add 10 µl of the antibiotic stock.
4. Pick a colony with the inoculation needle and transfer it to the liquid medium.
5. Incubate at 37 °C overnight, shaking at 250 rpm.

## NEBuilder HiFi DNA assembly

1. Set up assembly reactions on ice according to the table below:

   | Recommended amounts | | |
   | --- | --- | --- |
   |  | Assembly of 2-3 fragments | Assembly of 4-6 fragments |
   | Vector:insert ratio | 1:2 | 1:1 |
   | Fragments | 0.03-0.2 pmol | 0.2-0.5 pmol |
   | NEBuildier HiFi DNA assembly Master mix | 10 µl | 10 µl |
   | Milli-Q water | X µl | X µl |
   | **Final volume** | 20 µl | 20 µl |
2. Incubate at 50 °C for 15 minutes for 2-3 fragments; one hour for 4-6 fragment.
3. Store at -20 °C until use.

Protocol adapted from New England Biolabs (NEBuilder HiFi DNA assembly).

## PCR (Q5 HF)

1. Add the following components in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| Milli-Q water | X |
| Q5 High-Fidelity 2x Master Mix | 10 |
| 10 µM FW Primer | 2.5 |
| 10 µM RV Primer | 2.5 |
| 10 mM dNTPs | 1 |
| DNA Template (< 1,000 ng) | Y |
| Q5 High-Fidelity DNA Polymerase | 0.5 |
| **Final volume** | 50 |

2. Introduce the PCR tubes in the thermocycle and use the following PCR protocol:

| Step | Temperature (°C) | Time | #cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 seconds | 1 |
| Denaturation | 98 | 5-10 seconds | 25-35 |
| Annealing | 50-72 (based on Tm) | 10-30 seconds | 25-35 |
| Extension | 72 | 20-30 seconds/kb | 25-35 |
| Final Extension | 72 | 2 minutes | 1 |
| Hold | 12 | ∞ |  |

Protocol adapted from New England Biolabs (PCR Using Q5® High-Fidelity DNA Polymerase).

## PCR (Phusion HF)

1. Add the following components in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| Milli-Q water | X |
| 5X Phusion HF or GC Buffer | 10 |
| 10 µM FW Primer | 2.5 |
| 10 µM RV Primer | 2.5 |
| 10 mM dNTPs | 1 |
| DNA Template (< 250 ng) | Y |
| Phusion DNA Polymerase | 0.5 |
| **Final volume** | 50 |

2. Introduce the PCR tubes in the thermocycle and use the following PCR protocol:

| Step | Temperature (°C) | Time | #cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 seconds | 1 |
| Denaturation | 98 | 5-10 seconds | 25-35 |
| Annealing | 45-72 (based on Tm) | 10-30 seconds | 25-35 |
| Extension | 72 | 15-30 seconds/kb | 25-35 |
| Final Extension | 72 | 5-10 minutes | 1 |
| Hold | 12 | ∞ |  |

Protocol adapted from Thermo scientific (Phusion™ High–Fidelity DNA Polymerase).

## PCR & DNA Cleanup (NEB Monarch®)

1. Dilute your sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex.

| Sample type | Ratio binding buffer : Sample |
| --- | --- |
| dsDNA > 2kb (plasmids, gDNA) | 2:1 |
| dsDNA < 2kb (some amlicons, fragments) | 5:1 |

2. Insert a column into a collection tube and the load sample onto the column.
3. Spin for 1 minute, then discard the flow-through.
4. Re-insert the column into the collection tube. Add 200 μl of DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
5. Repeat step 4.
6. Transfer the column to a clean 1.5 ml microfuge tube.
7. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix.
8. Wait for 1 minute, then spin for 1 minute to elute the DNA.
9. Determine DNA concentration. Store at -20 °C.

Protocol adapted from New England Biolabs (Monarch® PCR & DNA Cleanup Kit - 5 μg).

## Plasmid isolation (Promega PureYield™ Plasmid Miniprep)

1. Transfer 1.5 ml of bacterial culture to sterile 1.5 ml microcentrifuge tube(s).
2. Centrifuge at max speed for 5 minutes. Discard supernatant. Resuspend the pellet in 600 µl of LB media.
3. Add 100 µl of Cell Lysis Buffer (blue), and mix by inverting the tube 6 times.
4. Add 350 µl of cold (4–8 °C) Neutralization Solution, and mix thoroughly by inverting.
5. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
6. Transfer the supernatant (~900 µl) to a PureYield™ Minicolumn without disturbing the cell debris pellet.
7. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
8. Discard the flow-through, and place the minicolumn into the same Collection Tube.
9. Add 200 µl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds.
10. Add 400 µl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.
11. Transfer the minicolumn to a clean 1.5 ml microcentrifuge tube, then add 20 µl of Milli-Q water directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
12. Centrifuge for 15 seconds to elute the plasmid DNA. Determine plasmid DNA concentration. Store at -20 °C.

Protocol adapted from Promega (PureYield™ Plasmid Miniprep System).

## RNA isolation (RNAeasy MiniElute)

1. Adjust the sample to a volume of 100 µl with Milli-Q water. Add 350 µl Buffer RLT, and mix well.
2. Add 250 µl of 96–100% ethanol. Mix well by pipetting.
3. Immediately transfer the sample (700 µl) to an RNeasy MinElute spin column placed in a 2 ml collection tube. Centrifuge for 15 seconds at 8000 x g (≥10,000 rpm). Discard the flow-through.
4. Place the RNeasy MinElute spin column in a new 2 ml collection tube and add 500 µl RPE buffer. Centrifuge for 15 seconds at 8000 x g.
5. Discard the flow-through and add 500 µl of 80% ethanol to the RNeasy MinElute spin column. Centrifuge for 2 minutes at 8000 x g.
6. Place the RNeasy MinElute spin column in a new 2 ml collection tube and centrifuge for 5 minutes at full speed.
7. Place the RNeasy MinElute spin column in a new 1.5 ml collection tube and add 14 µl RNase-free water. Centrifuge for 1 minute at full speed, your RNA is now in your tube.

Protocol adapted from Qiagen (RNeasy MinElute Cleanup Kit).

## Transformation of chemically competent cells (TOP10)

1. Prepare a heat block at 42 °C.
2. Take an aliquot of TOP10 competent cells (~ 50 µl) from storage at -80 ºC. Thaw on ice for 10-15 minutes.
3. Add 1-100 ng plasmid DNA to the 50 µl of competent cells. Mix by inverting the tube 4-5 times.
4. Incubate on ice for 15 minutes.
5. Heat shock at 42 °C for exactly 45 seconds.
6. Place on ice for 5 minutes.
7. Add 250 µl of SOC medium.
8. Incubate at 37 °C with shaking for 1 hour.
9. Plate 100 µl of the cells on a pre-warmed (at ~ 37 °C) LB agar plate with the desired antibiotic.
10. Incubate at 37 °C overnight. Alternatively, you can incubate over the weekend, leaving the plate on the bench.

DRIVER - *in vitro* evolution of aptazymes protocols

## Sequences: suffixes and prefixes, primers, libraries and references

All the sequences required to conduct DRIVER and the library preparation for sequencing can be found in the file below:

Click [here](https://static.igem.org/mediawiki/2021/b/b0/T--TUDelft--DRIVER_Sequences.pdf) to download the sequences pdf.

## DRIVER

The DRIVER protocol for cleavage and uncleavage rounds can be found in the following files:

Click to download the DRIVER protocol for [cleavage](https://static.igem.org/mediawiki/2021/8/80/T--TUDelft--DRIVER_Cleavage_NL.pdf) and [uncleavage](https://static.igem.org/mediawiki/2021/3/3c/T--TUDelft--DRIVER_Uncleavage_L.pdf) pdf.

In our experiment we ran four samples in parallel:

| Sample name | Library | Ligand in uncleavage rounds (final concentration) |
| --- | --- | --- |
| N30-F | T7\_W\_N30\_N6\_X | B9 (100 µM) |
| N30-V | T7\_W\_N30\_N6\_X | B1, B6, B12 (100 µM) and B2 (43.2 µM) |
| N60-F | T7\_W\_N60\_N8\_X | B9 (100 µM) |
| N60-V | T7\_W\_N60\_N8\_X | B1, B6, B12 (100 µM) and B2 (43.2 µM) |

The use of a positive and a negative control throughout the process is recommended. Our positive control was a fixed sequence with known cleavage properties (gB-Theo), and our negative control a reaction with no-template added:

| Control name | DNA template | Ligand in uncleavage rounds (final concentration) |
| --- | --- | --- |
| Positive control (PC) | gB-Theo | Theophylline (9.75 mM) |
| Negative control (NC) | None | Water |

The rounds’ order matrix can be found in the spreadsheet below. Starting with two cleavage rounds to remove the sequences that do not cleave. Followed by 60 rounds of cleavage and uncleavage in a 1:1 ratio. The required primers for each round are also specified in the spreadsheet.

Click [here](https://static.igem.org/mediawiki/2021/1/19/T--TUDelft--DRIVER_Rounds_Spreadsheet.pdf) to download the spreadsheet pdf.

When the rounds are performed without a liquid handling robot, we recommend to run a denaturing Urea-PAGE gel after every transcription. Check the Urea-PAGE protocol below. An agarose gel after PCR is also recommended in the early rounds. Check [general protocol](#General), DNA and RNA electrophoresis.

The source of the reagents used in this protocol can be found [here](https://static.igem.org/mediawiki/2021/d/d2/T--TUDelft--DRIVER_Reagents.pdf).

Moreover, we created a DRIVER troubleshooting, tips, and tricks guide, which can be found on our [contribution page](https://2021.igem.org/Team%3ATUDelft/Contribution).

## Library preparation for sequencing

We sequenced the N30-F and N30-V libraries after 37 rounds (intermediate) and after 62 rounds (final). Here, the final round is used as an example to explain the protocol. The same procedure can be followed for any intermediate round.

For each condition (N30-F final and N30-V final), two independent rounds are performed in the absence (cleavage) or presence (uncleavage) of ligand(s):

| Condition | Sample name | Previous round (PCR template) | Sequencing round | Ligand present | Start prefix |
| --- | --- | --- | --- | --- | --- |
| N30-F final | N30-F\_R63c | 62 | 63 cleavage | No | W |
|  | N30-F\_R63u | 62 | 63 uncleavage | Yes | W |
| N30-V final | N30-V\_R63c | 62 | 63 cleavage | No | W |
|  | N30-V\_R63u | 62 | 63 uncleavage | Yes | W |

Both cleavage and uncleavage rounds follow the DRIVER protocol as described above up to the PCR step. During PCR, both forward primers, BT1285p (W prefix) and BT1510p (Z prefix), are added to amplify the uncleaved components and the cleaved components, respectively. Moreover, at the PCR step, 4 distinct reference sequences similar to the library structure, each with either a W or Z prefix, are spiked-in at a fixed 1.4 pM concentration each. These references allow computation of absolute concentrations of each sequence with each prefix. Additionally, only 5 PCR cycles are to be performed. The PCR sheet in the DRIVER protocols can be substituted with the files below:

Click to download the PCR step sheet for
[cleavage](https://static.igem.org/mediawiki/2021/d/db/T--TUDelft--DRIVER_PCR_Sequencing_Cleavage_NL.pdf) and [uncleavage](https://static.igem.org/mediawiki/2021/7/74/T--TUDelft--DRIVER_PCR_Sequencing_Uncleavage_L.pdf) pdf.

### NGS barcode addition through HiFi PCR

1. Dilute each PCR product 10X by adding 225 μl of water, vortex briefly and spin down.
2. Take 100 μl of the diluted PCR products from step 1 and add 3 μl of RNase A. Incubate at 37 °C for 30 minutes to remove any possible RNA contamination.
3. Add the following components in a PCR tube:

| Component | Volume (µl) |
| --- | --- |
| Milli-Q water | 27 |
| Q5 High-Fidelity 2x Master Mix | 10 |
| 10 µM FW Primer (W1 or W2) | 2.5 |
| 10 µM FW Primer (Z1 or Z2) | 2.5 |
| 10 µM RV Primer (X1 or X2) | 2.5 |
| 10 mM dNTPs | 1 |
| DNA Template (after RNase A treatment) | 4 |
| Q5 High-Fidelity DNA Polymerase | 0.5 |
| **Final volume** | 50 |

The reverse primer X1 is used to barcode folate samples (N30-F\_R63c and N30-F\_R63u). The reverse primer X2 is used to barcode vitamin samples (N30-V\_R63c and N30-V\_R63u). The forward primers W1 and Z1 are used to barcode cleavage samples (N30-F\_R63c and N30-V\_R63c). The forward primers W2 and Z2 are used to barcode uncleavage samples (N30-F\_R63u and N30-V\_R63u).

Run 5 μl of the PCR products on an agarose gel to check whether the size of the amplicon is correct. For DNA electrophoresis, check [general protocol](#General), DNA and RNA electrophoresis.

### Sequencing Library Mixdown

1. Perform a clean-up of the PCR products. For this step, we recommend the PCR and DNA clean up kit 5 μg (Monarch®) from New England Biolabs. Check [general protocol](#General), DNA Cleanup (NEB Monarch®). Elute products in 25 μl of Milli-Q water.
2. Quantify products using Qubit® dsDNA HS Assay Kit. For DNA concentration determination using Qubit®, check [general protocol](#General), DNA concentration determination: Using Qubit® dsDNA HS Assay Kits.
3. Mix the four samples (N30-F\_R63c, N30-F\_R63u, N30-V\_R63c and N30-V\_R63u) in equimolar concentrations.
4. The sample can be sequenced on an Illumina platform. In our case, after the library mixdown, samples were sent to Eurofins Genomics. Sequencing adapters were added via ligation ([NGSelect Amplicons Adaptor Ligation](https://eurofinsgenomics.eu/en/next-generation-sequencing/ngs-build-your-own/ngselect-amplicons/ngselect-amplicons-adapter-ligation)). The sample was then sequenced using the Illumina NovaSeq sequencing technology, with 2 x150 bp paired-end reads.

## Characterization of aptazyme cleavage fractions through transcription and denaturing PAGE

1. Prepare the following reaction mixture:

| Component | Volume (µl) | Final concentration |
| --- | --- | --- |
| 10X RNA pol buffer | 2 | 1X |
| rNTPs (25 mM each) | 1.8 | 9 mM |
| DTT (500 mM) | 0.4 | 10 mM |
| SUPERase In (20 U/µl) | 1 | 1 U/µl |
| T7 RNA pol (50 U/µl) | 2 | 5 U/µl |
| DNA template | X | 10 nM |
| Ligand | Y | Y |
| Milli-Q water | Z |  |
| **Final volume** | 20 |  |

2. Incubate at 37 °C for 30 minutes.
3. Stop the transcription and cleavage reactions by adding EDTA to
   a final concentration of 4 mM.
4. Prepare samples, pre-run, run and stain gel as described in the Urea-PAGE protocol below.

## Urea-PAGE

### Gel casting

1. Mix the following components in a Duran bottle:

| Component | Volume (for 2 gels, 15 ml) | Final concentration |
| --- | --- | --- |
| Urea | 7.2 g (~ 5.5 ml) | 8 M |
| TBE 10X | 1.5 ml | 1X |
| Distilled water | 4.2 ml |  |
| Acrylamide:Bisacrylamide 29:1 (40%) | 3.75 ml | 10% |

2. Microwave at medium-low power for 15-20 seconds or until all the urea is dissolved. Do not overheat as cyanates may begin to form.
3. Cool down the mixture in the fridge. Ensure that the lid is closed and let it lay sideways on the bottom of the fridge for fast cooling.
4. While waiting for step 3, assemble the gel cassette.
5. Add 150 µl of 10% APS and 7.5 µl of TEMED and mix well. The final mixture has to be cold, otherwise it will polymerize in the bottle and you will not be able to pipette it.
6. Pour/pipette the gel (~ 6 ml per gel). Insert the comb quickly making sure there are no bubbles in the comb.
7. Leave the tip inside the Duran bottle. When the remainder of the solution has polymerized (~ 20-30 minutes), your gel will be ready.
8. Gels can be stored at 4 °C for up to 1 week.

### Pre-run

1. Assemble the gel and immerse in TBE running buffer.
2. Remove the comb and carefully rinse the wells with a pipette. You have to see how the urea comes out of the wells.
3. Pre-run the gel at 120 V for 30 minutes. During this time, prepare your samples as described below.
4. Rinse the wells again.

### Sample preparation

1. Mix 7.5 µl of RNA sample with 7.5 µl of 2X RNA loading buffer. Incubate at 95 °C for 10 minutes. Spin down before loading onto the gel. Prepare also the ssRNA ladder in the same way during this step.

### Run

1. Load the samples into the wells.
2. Run the gel at 220 V until the front goes away (~ 50 minutes).

### Gel staining and imaging

1. Immerse the gel in a SYBR Safe staining solution (50 µl SYBR Safe in 50 ml 1X TBE buffer) for 5-15 minutes. The staining solution can be reused, but it must be kept in total darkness to prevent degradation of SYBR Safe.
2. Visualize gel in a BioRad Imaging System.

## Vitamin stock solutions

1. Dissolve the required amount of vitamins in Milli-Q water according to the following table:

| Vitamin | Vitamin amount (mg) | Milli-Q water (ml) | Final concentration (µM) |
| --- | --- | --- | --- |
| Vitamin B1 (Thiamine pyrophosphate) | 5.3 | 10 | 2000 |
| Vitamin B2 (Riboflavin) | 2.7 | 40 | 180 |
| Vitamin B6 (Pyridoxal 5′-phosphate) | 3.4 | 10 | 2000 |
| Vitamin B9 (Folic acid) | 8.8 | 30 | 664.5 |
| Vitamin B12 (Cobalamin) | 6.8 | 2.5 | 2000 |

All vitamins were purchased from Sigma-Aldrich, except for riboflavin that was purchased from ThermoFisher.

2. To dissolve vitamin B9 (folic acid), adjust the pH of the solution to pH 8 using 1 M NaOH.
3. Store the solutions at 4 °C in dark nalgene bottles for up to 1 week.

Cell-free expression system protocols

## TnT® SP6 High-Yield Wheat Germ Protein Expression System (Promega)

1. Take TnT® SP6 High-Yield Wheat Germ Master Mix from storage at -80 ºC. Rapidly thaw the master mix on ice or by hand warming, and immediately place on ice.
2. After the master mix has thawed, gently mix several times by pipetting.
3. Assemble the following reaction components in a nuclease-free reaction vessel (e.g. 0.5 ml microcentrifuge tube):

| Component | Volume (µl) |
| --- | --- |
| TnT® SP6 High-Yield Wheat Germ Master Mix | 12 |
| DNA Template (BYDV vector\*) | X (0.5-5 nM) |
| Milli-Q water | Y |
| Theophylline | Z |
| **Final volume** | 20 |

\* BYDV vectors incorporate sequences from the barley yellow dwarf virus (BYDV) upstream and downstream of the protein-coding region of interest.

4. Mix gently after all components are added to the reaction tube. Place back on ice.
5. (Optional) Transfer the assembled reaction to a 384-well plate if measurements are to be taken in a plate reader.
6. Incubate the reaction at 25 °C for 2 hours.

Protocol adapted from Promega (TnT® SP6 High-Yield Wheat Germ Protein Expression System).

## myTXTL® T7 Expression Kit

1. Take the myTXTL LS70 Master Mix, the helper plasmid P70a-T7rnap and the positive control plasmid T7p14-deGFP from storage at -80 ºC. Rapidly thaw the master mix on ice or by hand warming, and immediately place on ice.
2. Vortex the myTXTL LS70 Master Mix for 2-3 seconds and briefly spin down. If any precipitate is visible hereafter, gently resuspend master mix solution about 10 times to ensure homogeneity. Avoid formation of bubbles and foam.
3. Assemble the following reaction components in a nuclease-free reaction vessel (e.g. 0.5 ml microcentrifuge tube):

| Component | Volume (µl) |
| --- | --- |
| Sigma 70 Master Mix | 15 |
| P70a-T7rnap HP (2.4 nM) | 0.83 |
| DNA Template | X (0.5-5 nM) |
| Milli-Q water | Y |
| **Final volume** | 20 |

4. Mix gently after all components are added to the reaction tube. Place back on ice.
5. (Optional) Transfer the assembled reaction to a 384-well plate if measurements are to be taken in a plate reader.
6. Incubate the reaction for up to 16 hours at 29 °C.

Protocol adapted from Arbor Biosciences (myTXTL® T7 Expression Kit).

## PURExpress® In Vitro Protein Synthesis Kit

1. Take solutions A and B from storage at -80 °C, and thaw on ice.
2. Pulse-spin in microfuge to collect solutions at the bottom of the tube. Certain components in Solution A may precipitate during storage. Be sure to mix it well by pipetting prior to assembling reactions. Do not vortex solution B.
3. Assemble the following reaction components in a nuclease-free reaction vessel (e.g. 0.5 ml microcentrifuge tube):

| Component | Volume (µl) |
| --- | --- |
| Solution A | 8 |
| Solution B\* | 6 |
| SUPERase•In™ RNase Inhibitor (20 U/μl) | 0.5 |
| CPRG (10.9 mg/ml)\*\* | 1.11 |
| DNA Template | X (0.5-5 nM) |
| Milli-Q water | Y |
| Theophylline | Z |
| **Final volume** | 20 |

\*Add Solution B to Solution A, do not dilute Solution B unbuffered

\*\*To prepare a 10.9 mg/ml CPRG stock solution, weigh 10.9 mg of CPRG and dissolve it in 1 ml of Milli-Q water. Make aliquots of the solution and store at -20 °C. When preparing a PURExpress® In Vitro Protein Synthesis reaction, thaw a CPRG aliquot on ice and wrap it in aluminum foil. Discard the remainder of the solution, do not reuse it.

4. Mix gently and pulse-spin in microfuge to collect mixture at the bottom of the tube.
5. (Optional) Transfer the assembled reaction to a 384-well plate if measurements are to be taken in a plate reader.
6. Incubate the reaction at 37 °C for 2 hours.

Protocol adapted from New England Biolabs (PURExpress® In Vitro Protein Synthesis Kit).

## PUREfrex® 2.0

1. Take solutions I, II and III from storage at -80 ºC, and thaw on ice.
2. Pulse-spin in microfuge to collect solutions at the bottom of the tube. Vortex solution I to ensure proper mixing. Gently mix solutions I and II by pipetting. Solutions II and III should not be vortexed.
3. Assemble the following reaction components in a nuclease-free reaction vessel (e.g. 0.5 ml microcentrifuge tube):

| Component | Volume (µl) |
| --- | --- |
| Solution I | 10 |
| Solution II | 1 |
| Solution III | 2 |
| SUPERase•In™ RNase Inhibitor (20 U/μl) | 0.5 |
| DNA Template | X (0.5-5 nM) |
| Milli-Q water | Y |
| Theophylline | Z |
| **Final volume** | 20 |

4. Mix gently and pulse-spin in microfuge to collect mixture at the bottom of the tube.
5. (Optional) Transfer the assembled reaction to a 384-well plate if measurements are to be taken in a plate reader.
6. Incubate the reaction at 37 °C for 2 hours.
7. If X-Gal is used as substrate, add it to a final concentration of 10 mg/ml. Let the reaction proceed overnight at room temperature.

Protocol adapted from GeneFrontier (PUREfrex®2.0).

## Cell-free system reactions in paper discs

### Paper discs preparation

1. Cut 4 mm paper discs (Whatman, 1442-042) using a hole puncher.
2. Place the discs in a glass petri dish.
3. Sterilize by autoclaving.

### Cell-free system reaction assembly

1. In sterile conditions, place a disc in the wall of a 1.5 ml microcentrifuge tube.
2. Assemble the following reaction components in a nuclease-free reaction vessel (e.g. 0.5 ml microcentrifuge tube).

Using PURExpress® In Vitro Protein Synthesis Kit:

| Component | Volume (µl) |
| --- | --- |
| Solution A | 3 |
| Solution B | 2.25 |
| SUPERase•In™ RNase Inhibitor (20 U/μl) | 0.2 |
| CPRG (10.9 mg/ml) | 0.412 |
| DNA Template | X (0.5-5 nM) |
| Milli-Q water | Y |
| **Final volume** | 7.5 |

Or using PUREfrex® 2.0:

| Component | Volume (µl) |
| --- | --- |
| Solution I | 3.75 |
| Solution II | 0.375 |
| Solution III | 0.75 |
| SUPERase•In™ RNase Inhibitor (20 U/μl) | 0.2 |
| CPRG (10.9 mg/ml) | 0.412 |
| DNA Template | X (0.5-5 nM) |
| Milli-Q water | Y |
| **Final volume** | 7.5 |

3. Carefully transfer the mixture to the paper disc.
4. Do not close the lid of the 1.5 ml microcentrifuge tube, but cover the tube with parafilm. Make holes in the parafilm lid. Wrap the tube in aluminum foil to avoid CPRG degradation.
5. Put all your 1.5 ml microcentrifuge tubes in a box and wrap the box in aluminum foil.

### Lyophilization

1. Freeze the samples at -80 °C for two hours.
2. Lyophilize the samples overnight according to manufacturer instructions (Alpha 1–4 LD Plus).
3. After lyophilization, keep the paper discs at room temperature until use.

### Rehydration of paper discs

1. Transfer the discs to a 96-well plate in sterile conditions.
2. Rehydrate the paper discs with the desired concentration of ligand or Milli-Q water to the original reaction volume (7.5 µl).
3. Incubate at 37 °C for 2 hours.

Hardware protocols

## Paper discs preparation

1. Cut 4 mm paper discs (Whatman, 1442-042) using a hole puncher.

## Food color preparation

1. Dilute the food colorant in 1.5 ml tubes with Milli-Q water to lower the concentration (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, concentrations normalized by the undiluted concentration).

## CPR preparation

1. Dilute the CPR in 1.5 ml tubes with Phosphate Buffered Saline (PBS) (10x) to lower the concentration (0.5 mM, 0.75 mM, 0.3 mM, 0.4 mM, 0.5mM, 0.6 m, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM).

## Loading on the Hardware

*The samples are either loaded on the test cassette of the hardware or on a 96-well plate for the plate reader.*

1. Clean the wells of the test cassette with Milli-Q-water if a disc with food colorant is loaded, and Phosphate Buffered Saline (PBS) (10x) if a disc with CPR is loaded.
2. Use a tweezer to load the 4 mm paper disc in the wells of the test cassette.
3. Pipette 5.5 µL of the sample onto the paper disc.
4. Slide the test cassette into the hardware.
5. Measure the light intensity for 60 seconds.

## Plate reader

*The samples are either loaded on the test cassette of the hardware or on a 96-well plate for the plate reader.*

1. Load the paper discs in a 96-well plate
2. Pipette 5.5 µL of the sample onto the paper disc.
3. Measure the absorbance of the paper discs at a wavelength of 574 nm, or the absorbance spectrum from 300 to 1000 nM.

Electrophoretic Mobility Shift Assay protocols

## Sequences: DNA & RNA oligos

Sequences were generated and provided by the Vilnius iGEM team 2021, using their surface interactions aptamer prediction software:

| Oligonucleotide | Description | Sequence |
| --- | --- | --- |
| RBP4\_ssD | 30 nucleotides ssDNA oligo predicted to bind to the human retinol binding protein 4 | GTTGATTGTTATGTTTAGTGACGGGTTCCC |
| Rand\_ssD | 30 nucleotides random ssDNA oligo used as a negative control for binding | AGGGTCACATGGGCGTTTGGCACTACCGAC |
| RBP4\_RNA | 30 nucleotides RNA oligo predicted to bind to the human retinol binding protein 4 | GUCCCCCGCCCGUGUCCCGCUAGCCCCGCG |
| Rand\_RNA | 30 nucleotides random RNA oligo used as a negative control for binding | CUGUUUUCGAAAUUACCCUUUAAGCGCGGG |

All oligonucleotides were synthesized by Integrated DNA Technologies.

## Working RNase free

1. Before starting to work: Wash hands and wrists thoroughly. Dry with a paper towel. Put gloves on.
2. Pick a designated area for RNase free work. Only materials involved in the RNase free experiment must be introduced in this area, after being treated for RNase free work.
3. Clear working space of all materials and equipment. Clean bench surface with ethanol 70% and a paper sheet. Afterwards clean the bench surface with RNAzap spray and paper sheet.
4. Clean pipettes stand with ethanol 70% and introduce it in the RNase free working space.
5. Wet a paper sheet with 70% ethanol and clean pipette with it. Spray RNAzap onto a new paper sheet and clean the pipette with it. Pipette can then be placed on the pipette holder.
   Repeat for every pipette using the same RNAzap paper sheet.
6. Clear the outer surface of every material and equipment to be directly handled and held while working with RNase sensitive samples, using a paper sheet sprayed with RNAzap. Place all clean material inside the RNase free zone within reaching distance.
7. Before beginning work: Change gloves and spray a small amount of RNAzap. Rub hands to cover the gloves surface on the RNAzap.
8. When working in the RNase free zone: Avoid talking or otherwise use a facemask.
9. Do not touch face, skin, clothes, or any surface nor materials outside of the RNase free zone or that have not been treated against RNases. If any contamination of the hands were to happen, clean gloves with RNAzap.

## Native PAGE

1. Mix the following components in a Duran bottle (for 2 gels, 15 ml).

| Component | Volume (ml) | Final concentration |
| --- | --- | --- |
| Acrylamide:Bisacrylamide 29:1 (40%) | 3.75 | 10% |
| TBE 10X | 1.5 | 1X |
| Distilled water | 9.6 |  |

2. Let the mixture cool down in the fridge in order to retard polymerization upon addition of the next components.
3. Add 150 µl of 10% APS and 7.5 µl of TEMED and mix well.
4. Rapidly swirl container to assure mixture of the components and immediately use 1 ml pipette to pour the mixture into the glass cassettes. Quickly insert the comb, making sure no bubbles remain under the comb.
5. Leave the tip inside the Duran bottle. When the remainder of the solution has polymerized (~ 20-30 minutes), your gel will be ready.

## Oligonucleotides visualization

### Sample preparation

1. Resuspend oligonucleotides in required buffer (usually TE) or in Milli-Q water, as specified by the manufacturer.
2. Prepare further dilutions of the oligonucleotides as required.
3. Prepare ssRNA or ssDNA ladders. For preparation of the ssRNA ladder, mix RNA ladder with 2X RNA loading buffer in a 1:1 ratio (final volume 4 µl). Incubate at 95 °C for 10 minutes. Cool on ice for 3 minutes. For preparation of oligo standards ladder dilute ladder 1:1 with 50% glycerol and incubate at 95 °C for 10 minutes. Cool on ice for 3 minutes.
4. Prepare samples for loading into gels by diluting with Milli-Q water to the required concentration. Keep samples on ice.

### Gel electrophoresis

1. Assemble gel electrophoresis casette and pre-run gel at 120 V for 20 minutes.
2. Dilute samples to a 1:1 ratio using 50% glycerol stock solution. If working with ssDNA ladder, dilute in the same way. Mix by vortex and spin down.
3. Load ssDNA or ssRNA ladder into the first well of the gel. Load 15 µl of each sample into the wells of the gel.
4. Run PAGE at 120 V for 50 minutes. Wait for the tracking dye to be ¾ in the gel.

### Gel staining and visualization

1. Recover gel and stain in SYBR Safe (50 µl SYBR Safe in 50 ml 1X TBE buffer) for approximately 15 minutes.
2. Visualize gel in a BioRad Imaging System.
3. Identify suitable concentration of oligonucleotides for proper visualization.

## EMSA

### Binding Buffer preparation

1. Prepare a binding buffer 5X stock as indicated in the following table

| Component | Volume (µl) | Final concentration |
| --- | --- | --- |
| PBS buffer 10X | 250 | 5X |
| MgCl2 1 M | 2.5 | 5 mM |
| KCl 1M | 30 | 60 mM |
| Glycerol 80% | 156.25 | 25% |
| Milli-Q | 61.25 | - |
| **Final volume** | 500 | - |

2. Aliquote into working samples and store at -20 °C.

### Sample preparation

1. Prepare a master mix as indicated in the following table

| Component | Volume (µl) 1 reaction | Volume (µl) 10 reactions | Final concentration |
| --- | --- | --- | --- |
| Binding buffer 5X | 2 | 15 | 1X |
| DTT 10 mM | 0.075 | 0.75 | 0.1mM |
| BSA 1 mg/ml | 0.075 | 0.75 | 0.01 mg/ml |
| **Final volume** | 1.65 | 16.5 | - |

2. In PCR tubes, add oligonucleotides (50 ng) and target protein in equimolar ratios (1:0, 2:0.5, 1:0.5, 1:1, and 1:2).
3. Fill up to 5.85 µl with Milli-Q water.
4. Add 1.65 µl of master mix to each of the tubes.
5. Incubate at 37 °C for 45 minutes.
6. Prepare ssRNA or ssDNA ladders. For preparation of the ssRNA ladder, mix RNA ladder with 2X RNA loading buffer in a 1:1 ratio (final volume 4 µl). Incubate at 95 °C for 10 minutes. Cool on ice for 3 minutes. For preparation of oligo standards ladder dilute ladder 1:1 with 50% glycerol and incubate at 95 °C for 10 minutes. Cool on ice for 3 minutes.

### Gel electrophoresis

1. Assemble gel electrophoresis casette and pre-run gel at 120 V for 20 minutes.
2. Dilute samples to a 1:1 ratio using 50% glycerol stock solution. If working with ssDNA ladder, dilute in the same way. Mix by vortex and spin down.
3. Load ssDNA or ssRNA ladder into the first well of the gel. Load 15 µl of each sample into the wells of the gel.
4. Run PAGE at 120 V for 40 minutes. Wait for the guiding dye to be ¾ in the gel.

### Gel staining and visualization

1. Stain in SYBR Safe (50 µl SYBR Safe in 50 ml 1X TBE buffer) for approximately 15 minutes.
2. Visualize gel in a BioRad Imaging System.

Microfluidic chip printing protocols

### Printing microfluidic channels

1. Create your desired shape template. To this, you can use, for example, Microsoft powerpoint.
2. Print out the template on A4 paper.
3. Use masking tape to attach 5 chromatography papers (Whatman qualitative filter paper, Grade 1 175x100 mm) to A4 template.
4. Print microfluidic channels on the Whatman paper.
5. Print settings:

   * Resolution: 1200 dots per inch (dpi)
   * Printer: Xerox AltaLink C8055
6. Remove the masking tape.

### Impregnating the ink

1. Preheat oven at 165 °C.
2. Put the papers in a glass container that can withstand 165 °C.
3. Keep the container in the oven for 15 minutes.
4. Carefully take it out of the oven and let it cool.

### Testing the chip

Pipette 40 µl of food coloring onto the microfluidic paper chip.

## A big thank you to our sponsors!

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# Team:TUDelft/Results

AptaVita

AptaVita

[![homelogo](https://static.igem.org/mediawiki/2021/c/c9/T--TUDelft--navbar_full%2BTU.png "homelogo")](https://2021.igem.org/Team%3ATUDelft)

Team

[Team Members](https://2021.igem.org/Team%3ATUDelft/Team)
[Collaborations](https://2021.igem.org/Team%3ATUDelft/Collaborations)
[Attributions](https://2021.igem.org/Team%3ATUDelft/Attributions)
[Sponsors](https://2021.igem.org/Team%3ATUDelft/Sponsors)

Project

[Project Description](https://2021.igem.org/Team%3ATUDelft/Description)
[Design](https://2021.igem.org/Team%3ATUDelft/Design)
[Engineering Success](https://2021.igem.org/Team%3ATUDelft/Engineering)
[Results](https://2021.igem.org/Team%3ATUDelft/Results)
[Hardware](https://2021.igem.org/Team%3ATUDelft/Hardware)
[Contribution](https://2021.igem.org/Team%3ATUDelft/Contribution)
[Sequencing analysis](https://2021.igem.org/Team%3ATUDelft/Software)
[Partnership](https://2021.igem.org/Team%3ATUDelft/Partnership)
[Sustainable Development](https://2021.igem.org/Team%3ATUDelft/Sustainable)

Lab

[Protocols](https://2021.igem.org/Team%3ATUDelft/Experiments)
[Notebook](https://2021.igem.org/Team%3ATUDelft/Notebook)
[Parts](https://2021.igem.org/Team%3ATUDelft/Parts)

Model

[Model](https://2021.igem.org/Team%3ATUDelft/Model)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP)
[Education & Public Engagement](https://2021.igem.org/Team%3ATUDelft/Communication)
[Proposed Implementation](https://2021.igem.org/Team%3ATUDelft/Implementation)
[Safety](https://2021.igem.org/Team%3ATUDelft/Safety)
[Entrepreneurship](https://2021.igem.org/Team%3ATUDelft/Entrepreneurship)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3806)
[Medal Criteria](https://2021.igem.org/Team%3ATUDelft/Achievements)
[Awards and Nominations](https://2021.igem.org/Team%3ATUDelft/Awards)

# Results

On this page, we present the results of our experiments, including engineering of the vitamin-binding aptazyme through *in vitro* evolution, cell-free expression of our genetic construct, and the absorbance measurements conducted with our dedicated read-out device.

* [AptaVita](#Intro)
* [Engineering aptazymes for vitamin detection](#DRIVER)
* [Cell-free expression system](#Expression)
* [Hardware](#Hardware)
* [References](#references)

## AptaVita

AptaVita aims to develop a quantitative, accessible, and modular biosensor that is able to detect vitamin deficiencies from blood samples. Throughout our project, we engineered our biosensor consisting of a ligand-regulated self-cleaving aptazyme that is fused to a reporter gene to give a colorimetric read-out of the vitamin concentration. This genetic construct was expressed in a cell-free system on paper support and the vitamin concentration was quantitatively analyzed by our dedicated hardware through absorbance measurements.

## Engineering aptazymes for vitamin detection

### Introduction

To construct our biosensor, we first engineered ligand-regulated self-cleaving aptazymes that detect vitamins through a novel iterative *in vitro* evolutionary method, [*De novo* Rapid *In Vitro* Evolution of RNA biosensors (DRIVER)](https://2021.igem.org/Team%3ATUDelft/Design#DRIVER) [[1]](#references). Each round of DRIVER consists of a transcription, reverse transcription, ligation, and PCR steps. For these evolutionary experiments, we alternated the transcription conditions between the absence and presence of ligands. This results in selection of aptazymes that selectively self-cleave in the absence of the ligands, and remain uncleaved in their presence. We chose folate (vitamin B9), thiamine pyrophosphate (B1), riboflavin (B2), pyridoxal 5′-phosphate (B6), and cobalamin (B12) as our target ligands.

### Setup of DRIVER protocol

#### Validation of transcription protocol

To validate the first step of our DRIVER protocol, we decided to test an already known theophylline-binding aptazyme, gB-Theo([BBa\_K3806008](http://parts.igem.org/Part%3ABBa_K3806008)) This 109-bp aptazyme binds selectively to theophylline and was discovered by Townshend *et al.* [[1]](#references), the same authors that developed the DRIVER method. We run a Urea-PAGE gel to visualize the transcription products of the gB-Theo aptazyme with and without the addition of theophylline at different concentrations. The bands corresponding to the uncleaved and cleaved products of the theophylline aptazyme are expected to be located at 109 bp and 89 bp, respectively. ImageJ was used to quantify the intensities (I) of the gel bands, and the cleavage fraction (f) was determined by:

![](https://static.igem.org/mediawiki/2021/9/9e/T--TUDelft--Gel_Fold_Change_LaTeX_Formula.PNG)

We observed that the cleavage fraction decreases when increasing theophylline concentration: 71 % at 0 mM theophylline (Fig. 1, lane 3), 39 % at 0.5 mM theophylline (lane 4), and 4 % at 10 mM theophylline (lane 5). With this experiment, we successfully validated our transcription protocol and confirmed the ligand-dependent cleavage activity of the gB-Theo aptazyme [[1]](#references). Therefore, we used gB-Theo as a positive control throughout our entire DRIVER experiment.

![theophylline aptazyme cleavage characterization](https://static.igem.org/mediawiki/2021/9/9a/T--TUDelft--R1.png "theophylline aptazyme cleavage characterization")

*Fig. 1 Urea-PAGE gel for characterization of theophylline aptazyme cleavage fractions following a co-transcriptional cleavage assay.* Lanes: (Ladder) denatured dsRNA ladder, (1) Negative control: no DNA template. (2) gB-Theo DNA template (without T7 RNA polymerase added to the transcription reaction), transcription of gB-Theo with (3) 0 mM theophylline, (4) 0.5 mM theophylline, (5) 10 mM theophylline.

#### Compatibility of target ligand solution in transcription

To engineer aptazymes for vitamin detection, target ligands are supplemented to the transcription reaction. We learned from our engineering cycle that potential interference of the ligand addition with transcription can be prevented by finding a compromise between ligand solubility and pH of the solution. The establishment of the reaction conditions for the transcription step is explained in detail on our [Engineering Success](https://2021.igem.org/Team%3ATUDelft/Engineering) page. In brief, to find this compromise, we prepared folate solutions at different pH and supplemented the transcription reaction, we used gB-Theo as the DNA template and ran a Urea-PAGE gel to visualize the transcription products.

We found that the addition of folate solution adjusted to a pH range of 8 to 9 does not affect transcription. This can be observed in Fig. 2 (lanes 3 to 6) as the intensity of the transcript products was comparable to that of the positive gB-Theo control (lane 7, no folate solution added).

![influence folate pH](https://static.igem.org/mediawiki/2021/e/e8/T--TUDelft--R2.png "influence folate pH")

*Fig. 2 Urea-PAGE gel to assess the influence of the pH of folate solutions on transcription.* Lanes: (Ladder) denatured dsRNA ladder, (1) Negative control: no DNA template. (2) folate solution (pH 14.0), (3) folate solution (pH 9), (4) folate solution (pH 8.5), (5) folate solution (pH 8), (6) folate solution (pH 8) and 10 mM theophylline, and (7) Positive control: without ligand. gB-Theo was used as the DNA template for lanes 2 to 7. The pH of the folate solution was adjusted using NaOH.

The pH of the vitamin mixture (B1, B2, B6 and B12) was already within a range compatible with transcription and therefore used to perform DRIVER rounds.

### DRIVER - *in vitro* evolution of aptazymes

As the transcription protocol was validated, also confirming its compatibility with target ligands, we started the *in vitro* selection rounds using the DRIVER method. Two high-diversity libraries of approximately 1012 potential biosensor sequences were used as inputs to DRIVER. The first library consisted of sequences with a large loop of 30 random nucleotides (N30) and the second library consisted of sequences with a large loop of 60 random nucleotides (N60). Throughout the aptazyme engineering process, two ligand(s) conditions were used: a folate solution and a mixture of vitamin B1, B2, B6, and B12. Combination of the two libraries and the two ligand conditions results in a total of four samples (N30-F, N30-V, N60-F, and N60-V). These four samples were used alongside a positive (gB-Theo) and negative (no DNA template) control on every evolution round. For further explanation on these design choices, visit our [Design](https://2021.igem.org/Team%3ATUDelft/Design) page.

As a first step on our evolution cycles, we run two cleavage selection rounds without the addition of ligands, to enrich the aptazymes that are able to cleave. We then run an Urea-PAGE after each round to visualize the transcript products. After the transcription step of the second round, transcription products could be observed (Fig. 3), suggesting that all the steps of the first round: transcription, RT, ligation, and PCR, were successful. Additionally, by comparing the two gels, it can be seen that in the second round, there is a slight enrichment in the cleavage fraction as the lower band intensifies (lanes 7 and 8 vs lanes 3 and 4). From these results, we conclude that the DRIVER method successfully enriched self-cleaving sequences during the first cleavage selection round.

![round 1 and 2](https://static.igem.org/mediawiki/2021/b/ba/T--TUDelft--R3.png "round 1 and 2")

*Fig. 3 Urea-PAGE gel with the transcription products of round 1 (left) and 2 (right).* Lanes: (Ladder) ssRNA ladder, (1) Negative control: no DNA template. (2) gB-Theo, (3) potential aptazymes with a large loop of 30 random nucleotides (N30), (4) potential aptazymes with a large loop of 60 random nucleotides (N60). No ligands were added to enrich self-cleaving aptazymes.

We continued running DRIVER for 62 rounds, altering between +ligand (uncleavage) and -ligand (cleavage) rounds. With this, we enriched the population with the responsive aptazymes to our target vitamins. We run an Urea-PAGE gel after each round to confirm the transcription products. The intermediate results of the selected Urea-PAGE gels can be found in the *DRIVER intermediate rounds-supplementary data* below. One should notice that Urea-PAGE gels display high variability between rounds due to handling variation, thus the DRIVER method is highly suggested to be robot-automated. Yet, the use of Urea-PAGE was an important tool through the whole of our evolutionary process, which allowed us to follow the progression of the rounds. For instance, the N60 libraries were progressively lost, and no transcript products could be seen after 43 rounds of selection. This outcome is coherent with the results in the study of Townshend et al [[1]](#references), where all but one of the aptazymes discovered presented a larger loop with less than 60 nucleotides. We hypothesize that the increase in the length of the large loop results in a large change of the aptazyme structure that hinders aptazyme self-cleavage, and therefore the selection process. Considering the aforementioned, we put our expectations on the N30 libraries and proceeded with NGS.

Supplementary figures - DRIVER

![round 13 and 14](https://static.igem.org/mediawiki/2021/0/05/T--TUDelft--RS1.png "round 13 and 14 style=")

*Supplementary Fig. 1 Urea-PAGE gel with transcription products of round 13 (+ ligand, left) and 14 (- ligand, right).* (Ladder) ssRNA ladder, (NC) Negative control: no DNA template. (gB-Theo) gB-Theo +/- theophylline, (N30-F) aptazymes with a large loop of 30 random nucleotides +/- folate, (N30-V) aptazymes with a large loop of 30 random nucleotides +/- vitamin B1, B2, B6, and B12, (N60-F) aptazymes with a large loop of 60 random nucleotides +/- folate, (N60-V) aptazymes with a large loop of 60 random nucleotides +/- vitamin B1, B2, B6, and B12. The NC lane contains some contamination of RNA molecules. Accumulation of residual primers is seen at around 50 bp.

![round 36 and 37](https://static.igem.org/mediawiki/2021/4/44/T--TUDelft--RS2.png "round 36 and 37")

*Supplementary Fig. 2 Urea-PAGE gel with transcription products of round 36 (- ligand, left) and 37 (+ ligand, right).* (Ladder) ssRNA ladder, (NC) Negative control: no DNA template. (gB-Theo) gB-Theo -/+ theophylline, (N30-F) aptazymes with a large loop of 30 random nucleotides -/+ folate, (N30-V) aptazymes with a large loop of 30 random nucleotides -/+ vitamin B1, B2, B6, and B12, (N60-F) aptazymes with a large loop of 60 random nucleotides -/+ folate, (N60-V) aptazymes with a large loop of 60 random nucleotides -/+ vit B1, 2, 6, and 12. The NC lane contains contamination of RNA molecules. Accumulation of residual primers are seen at around 65 and 50 bp.

![round 57 and 58](https://static.igem.org/mediawiki/2021/e/ec/T--TUDelft--RS3.png "round 57 and 58")

*Supplementary Fig. 3 Urea-PAGE gel with transcription products of round 57 (+ ligand, left) and 58 (- ligand, right).* (Ladder) ssRNA ladder, (NC) Negative control: no DNA template. (gB-Theo) gB-Theo +/- theophylline, (N30-F) aptazymes with a large loop of 30 random nucleotides +/- folate, (N30-V) aptazymes with a large loop of 30 random nucleotides +/- vitamin B1, B2, B6, and B12. Reactions for N60 libraries were not continued as aptazyme sequences were lost after round 43. A new NC and gB-Theo was taken from round 52. Accumulation of residual primers are seen at around 65 and 50 bp in reactions with N30-F and N30-V for both rounds.

### Library preparation for next-generation sequencing

To identify enriched sequences that exhibit a higher cleavage fraction in the absence of the target vitamins and lower fraction in their presence, DRIVER-evolved libraries should be screened using [next-generation sequencing (NGS)](https://2021.igem.org/Team%3ATUDelft/Design#NGS). Before NGS, libraries were prepared using the product of round 62 to run two distinct rounds: in the presence and absence of the ligands of interest (Fig. 4). For each condition, cleaved and uncleaved populations were amplified enabling computation of the cleavage fraction for each sequence in the mixture after NGS.

![round 63](https://static.igem.org/mediawiki/2021/3/38/T--TUDelft--R4.png "round 63")

*Fig. 4 Urea-PAGE gel with transcription products of round 63 without ligand (left) and with ligand (right).* (Ladder) ssRNA ladder, (NC) Negative control: no DNA template. (gB-Theo) gB-Theo +/- theophylline, (N30-F) aptazymes with a large loop of 30 random nucleotides +/- folate, (N30-V) aptazymes with a large loop of 30 random nucleotides +/- vit B1, 2, 6, and 12. Reactions for N60 libraries were not continued as no transcript product could be visualized in Urea-PAGE after round 43. The NC lanes contain contaminations of RNA molecules. Accumulation of residual primers are seen at around 65 and 50 bp.

### Aptazyme hit identification through next-generation sequencing

The NGS data analysis was performed with our sequence analysis pipeline, developed based on the work of Townshend *et al.* [[1]](#references). For details on the development of this sequencing pipeline visit our [Software](https://2021.igem.org/Team%3ATUDelft/Software) page. Our sequence analysis pipeline analyzes the sequences based on their ability to remain uncleaved in the presence of the target vitamin and to cleave in its absence. A scatter plot is generated to portray how the population of potential aptazyme sequences distribute based on the fraction that underwent cleavage. Potential aptazyme candidates are expected to be located in the bottom right quadrant of the scatter plot, displaying a large cleavage fraction in the absence of the targets, while simultaneously having a low cleavage fraction in their presence. On the other hand, ligand-insensitive sequences are expected to have the same cleavage fraction in the presence and absence of the ligand, and therefore be located along the diagonal of the scatter plot.

The scatter plots for both the folate and vitamin mixture libraries are shown in Fig. 5. As can be noted from this figure, most of the sequences do not exhibit a cleaved fraction in the presence of their ligand and are located on the x-axis rather than on the trend line (dashed line). We hypothesize this could be due to low efficiency in prefix replacement during the NGS library preparation in the presence of the ligand. Nevertheless, a high number of sequences are located in the bottom right quadrant of the scatter plots, which suggests the possible presence of potential aptazyme sequences.

![](https://static.igem.org/mediawiki/2021/a/a6/T--TUDelft--Sequence_Analysis_Cleavage_Fractions.svg)

Fig. 5 *Fraction cleaved in the absence (x-axis) and presence (y-axis) of the target ligands for potential aptazymes in a DRIVER enriched library after 63 rounds.* Only sequences with a read count of 25 or higher were considered as potential aptazymes and used in the analysis. The dashed line in magenta, represents the expected trendline of the library. (A) Folate library. The library contained 804 potential aptazyme sequences, of which 48 showed a fold change of cleavage. (B) Vitamin mix library. The library contained 1152 potential aptazyme sequences, of which 78 showed a fold change of cleavage.

Out of all the potential aptazyme candidates, we selected two sequences from the folate library and three from the vitamin mixture library as the five sequences to further investigate. We based this selection on two criteria: *(i)* the **fold change** of cleavage between – and + ligand conditions, which should be ideally equal or higher than 2 [[1]](#references), and *(ii)* the preservation of the initial **aptazyme’s structure**. The first candidate from the folate library presents a 2.56 fold change. Meanwhile, the rest of the five candidate sequences display a fold change below 2. These moderate fold changes values could be explained by the limited number of iterative rounds we performed, 63 rounds, close to the minimum of 57 rounds that was required to find a functional aptazyme in the study of Townshend et al [[1]](#references). For this reason, we hypothesize that our candidate sequences present a low affinity for the ligand, which is reflected in the fold change. For all candidates, a qualitative and quantitative characterization is required to validate their nature as aptazymes.

*Tab. 1 Identified potential aptazyme sequences. Sequences were selected based on their fold change and length.* The sequences are reported with the correspondent library, fold change, and the p-value.

| ID | Sequence | Library | Fold change | P-value |
| --- | --- | --- | --- | --- |
| A | GCTGTCACAGGTCTTCTGTCTGACGAGTCCTTGGCCGGACGAAACAGC | Folate | 2.56 | 1.37 · 10-13 |
| B | GCTGTCACCGGAAATCTTTTTGTACGCTACGAGTCACCGTTATCCGGTCTGAAGAGTCCATCGGTGGGACGAAACAGC | Folate | 1.48 | 9.57 · 10-8 |
| C | GCCTGTCATCGGATTCCATCCGGTCTGACGAGTCCTTGGCGGGACGAAACAGC | Vitamins | 1.88 | 1.68 · 10-16 |
| D | GCTGTCACCGGAAGTCTTTTTGTACGCTACGAGTCACCGTTATCCGGTCTGAAGAGTCCATCGGTGGGACGAAACAGC | Vitamins | 1.67 | 8.75 · 10-49 |
| E | GCTGTCACCGGAGTATTCTTGTTTGCTAATTGCGTCATCCGTTCCGGTCTGATGAGTCCAGTGGGGGACGAAACAGC | Vitamins | 1.63 | 7.87 · 10-12 |

Supplementary figures - sequencing

![read counts](https://static.igem.org/mediawiki/2021/8/86/T--TUDelft--Sequencing_Normalized_Read_Counts.svg "read counts")

*Supplementary Fig. 4 Sequence read counts normalized to reference read counts (y-axis) for the conditions + ligand and -ligand (x-axis) of the promising biosensor sequences shown in Tab. 1.* Reference read counts correspond to sequences that were added during the NGS preparation step at a known concentration, hence allowing for normalization of differences in amplification.

### Achievements

* DRIVER reaction conditions were established and 63 rounds were reached.
* A high-throughput assay based on next-generation sequencing was used to characterize enriched sequences in the library mixtures. Five potential aptazyme sequences were selected based on their fold change of cleavage between - and + ligand conditions, and the conservation of the aptazyme structure.

### Future prospects

#### Characterization of the potential vitamin-sensitive aptazymes

Validation of the NGS results is still necessary as this could not yet be performed. We suggest performing an Urea-PAGE assay in the absence and presence of the ligands for all the selected sequences. Aptazymes evolved using the vitamin mixture should be tested for each of the vitamins in the mixture, independently. Binding affinity can be further validated using a surface plasmon resonance assay as described by Townshend *et al.* in the original DRIVER manuscript [[1]](#references). Moreover, the binding selectivity of the aptamer sequences can be challenged by including structurally related molecules to the vitamin mixture. The chosen aptazymes should not respond to these analog molecules.

#### Improvement of aptazyme binding affinity

Based on the results obtained from these previous suggested experiments, additional evolution rounds can be performed to increase the sensitivity towards the target ligands. The ratio of cleavage and uncleavage selection rounds can be adjusted depending on the final application of the aptazyme. For instance, repeating two cleavage rounds after one uncleavage round can be done to bias selection towards producing sensors with higher cleavage fractions.

### Partnership iGEM Vilnius-Lithuania 2021

The goal of our project is the creation of an accessible, quantitative, and modular rapid diagnostic test capable of diagnosing the diversity of vitamin deficiencies. Our project currently targets water-soluble vitamins: B1, B2, B6, B9, and B12. Yet, new biosensors are required for vitamins A, C, D, E, and K, in order to include the detection of these protein-bound, non-water-soluble vitamins in our test. We partnered with the iGEM Vilnius-Lithuania 2021 team, who created an aptamer prediction software, to generate sequences with potential affinity for vitamin A’s carrier protein *retinol binding protein 4*. We used electrophoretic mobility shift assays (EMSA) under physiological compatible conditions in order to evaluate the binding capability of potential ssDNA and RNA aptamer sequences.

Molecular dynamic simulations and EMSA results

Our partnership with iGEM Vilnius-Lithuania 2021 provided us the opportunity to deepen into the modular aspect of our test, by working together in the development of aptamer sequences with affinity to the vitamin A carrier *retinol binding protein 4,* (RBP4). By experimentally validating the capability of their predictive software, we would be able to include the detection of protein-bound, non-water-soluble vitamins, in our diagnostic test. For them, our experimental results could validate their predictive software and generate experimental data on affinity for future improvements.

The Vilnius team modified their software in order to be able to generate RNA aptamer predictions, and fed it with the crystallographic structure of the human RBP4. They generated both ssDNA and RNA aptamers for us to experimentally validate. For this, we used an electrophoretic mobility shift assay to potentially demonstrate and quantify the affinity of these sequences for RBP4.

#### Computational analysis of RBP4 apo- and holo-proteins

Before conducting affinity experiments, we performed a 50-ns molecular dynamic (MD) simulation of our protein in NAMD [[2]](#references). We addressed two potential limitations:

1. Aptamers were predicted for the apo-RBP4 form. ​​Since we are interested in the holo-RBP4 form of the protein (vitamin bound), can an apo-form binding aptamer also bind to the holo-form?
2. Prediction is based on x-ray crystallography structures of the target protein. Crystallographic structures reveal only a static picture of the protein and can impose non-realistic physicochemical conditions. We therefore asked whether the protein allows for a proper aptamer binding under more realistic biological conditions?

![RMSD](https://static.igem.org/mediawiki/2021/a/a8/T--TUDelft--partnershipMD.svg "RMSD")

*Supplementary Fig. 5 (A) RMSD values of non-hydrogen atoms (backbone and side-chain) of the apo- and holo-forms of the human RBP4 protein in 50-ns MD simulations. (B) Structural superposition of apo- and holo-forms of the RBP4 protein after 50-ns MD simulations.*

Results showed very low mobility of both the apo- and holo-protein forms. Supplementary Fig. 5A shows the root mean square distances (RMSD) between the initial structural conformation and the succeeding frames of the simulation. For both the apo- and holo-forms the average RMSD was below 2 Å, 1.301 ± 0.195 Å, and 1.106 ± 0.195 Å, respectively. These low values confirmed the low protein mobility in any of their forms, suggesting that the protein-aptamer interactions computed using the crystallographic structure of the RBP4 protein, would probably not be affected by the protein mobility. Additionally, Supplementary Fig. 5B shows the superposition of the apo- and holo-RBP4 structures after the simulation, the RMSD computed was 1.56 Å (< 2 Å), supporting that the binding of vitamin A has a low impact on the structure of RBP4. This suggests that the aptamers generated or experimentally confirmed to bind to the apo-form could potentially form stable interactions with the retinol-bound form of the protein.

#### EMSA

In total, four 30 nucleotides sequences were computed and synthesized: a random ssDNA, a random RNA, a potential RBP4 ssDNA aptamer, and a potential RBP4 RNA aptamer. Random sequences were used as controls to show that aptamer-target interactions are sequence-specific. Incubation was carried at 37 °C for 40 minutes in a binding buffer formulated to mimic physiological ion composition. Details of our EMSA protocol can be consulted in our [Experiment & Protocol](https://2021.igem.org/Team%3ATUDelft/Experiments) page and the [Notebook](https://2021.igem.org/Team%3ATUDelft/Notebook).

Native polyacrylamide gel shows no apparent binding of ssDNA aptamer sequence for RBP4. Supplementary Fig. 6 shows the resulting native polyacrylamide gel for ssDNA sequences. Lanes 4 and 5 show the expected migration patterns for the free RBP4\_ssDNA and rand\_ssDNA oligos, in the absence of target protein. For all samples no shift in the migration pattern is observed when compared to the free oligo, indicating the lack of binding affinity between RBP4 and the potential aptamer sequences under the used conditions. The same results are seen for random\_RBP4 sequences.

![EMSA ssDNA](https://static.igem.org/mediawiki/2021/c/c2/T--TUDelft--EMSA_ssDNA.png "EMSA ssDNA")

*Supplementary Fig. 6 Fig. 4 Native-polyacrylamide gel of the EMSA conducted with ssDNA and RBP4 at different oligo:protein molar ratios. (Ladder) ssDNA oligo ladder, (NC) Negative control: no oligos nor target protein, (tracking oligos) show migration of free oligos through gel, (RPB4\_ssDNA) potential RBP4 ssDNA aptamers, (rand\_ssDNA) random ssDNA oligos. Lane 3 was skipped due to overflow of sample from lane 2. Lane 6 was used as a spacer. All ratios refer to RBP4\_ssDNA oligos:protein except for 1rand:1, where it refers to rand\_ssDNA. Ratios based on an oligo sample of 50 ng.*

Similar results were observed for the EMSA of RNA sequences in Supplementary Fig. 8. All samples migrated below the 50 nucleotides marker, with no visible shift for any of the ratios. Lane 3 and 4, containing rand\_RNA oligos, are barely perceptible. We attribute this to improper homogenization of the oligos before addition to the reaction mix. These results show that under the used conditions, the potential RNA aptamer sequences displayed no affinity for RBP4.

![EMSA RNA](https://static.igem.org/mediawiki/2021/7/7c/T--TUDelft--EMSA_RNA.png "EMSA RNA")

*Supplementary Fig. 7 Native-polyacrylamide gel of the EMSA conducted with RNA and RBP4 at different oligos:protein molar ratios. (Ladder) Low Range ssRNA ladder, (NC) Negative control: no oligos and no protein, (tracking oligos) show migration of free oligos through gel, (RPB4\_RNA) potential RBP4 RNA aptamers, (rand\_RNA) random RNA oligos. All ratios refer to RBP4 RNA oligos:protein ratios except for 1rand:1, where it refers to rand\_RNA. Ratios based on an oligo sample of 50 ng.*

#### Future prospects

The desired aptamer:protein binding interaction was not observed through EMSA’s at the used conditions. The EMSA methodology is highly protein and sequence dependent. As such, many physicochemical variables can have a strong impact on the experimental outcome. Therefore, different binding buffer compositions, oligos:protein ratios, incubation times and temperatures, and running times and voltages should be explored. During the development of the partnership, key limitations and improvement opportunities were identified for the predictive software of the iGEM Vilnius-Lithuania team. For more details on this and how our partnership developed through the extent of the iGEM competition, visit our [Partnership](https://2021.igem.org/Team%3ATUDelft/Partnership) page.

## Aptazyme-regulated reporter gene expression using a cell-free system on paper support

### Introduction

In keeping with our goal of making AptaVita an accessible and low-resource vitamin detection sensor, we engineered a genetic circuit to couple ligand-regulated cleavage of the aptazyme to a colorimetric output. A detailed explanation of the mechanism behind this genetic circuit can be found in our [Design](https://2021.igem.org/Team%3ATUDelft/Design) page. In short, the *lacZ* reporter gene, encoding for the β-galactosidase, was fused to an aptazyme sequence. Self-cleavage of the aptazyme in the absence of the ligand results in liberation of the ribosomal binding site (RBS), allowing for translation initiation of the β-galactosidase protein, which catalyzes the colorimetric substrate conversion of CPRG (yellow) to CPR (red). In the presence of the ligand, the aptazyme is stabilized preventing self-cleavage, the RBS is sequestered and translation is prohibited. As a proof-of-concept, we performed experiments to test the aptazyme-regulated *lacZ* expression in a cell-free system by using a known theophylline binding aptazyme [[1]](#references), which can eventually be replaced by the vitamin-responsive aptazymes engineered through DRIVER.

### Discovery of the suitable cell-free system for AptaVita First of all, we identified the appropriate cell-free system for our desired substrate, CPRG. After several iterations of the engineering cycle, we found that the PUREexpress cell-free system is compatible with our envisioned CPRG colorimetric read-out. Throughout the engineering cycle of finding this compatible cell-free/substrate combination, we also learned that PUREfrex2.0, another PURE expression system, is incompatible with CPRG because of undesired side reactions that degraded the substrate even without the presence of the construct. Nonetheless, we also discovered that PUREfrex2.0 can be used in combination with an alternative β-galactosidase substrate X-gal. In this results page, some of the experiments were performed using PUREfrex2.0 and X-gal because of our limited resources in PUREexpress. For more information about the above mentioned engineering cycle visit our [Engineering success](https://2021.igem.org/Team%3ATUDelft/Engineering) page. Enhancement of the biosensor performance through a new genetic constructComparing 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](http://parts.igem.org/Part%3ABBa_K3806014%20)) to a control part without an aptazyme switch. We used the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_I732017%20%20) part from the iGEM repository containing 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. The image shown in Fig. 6A, was taken after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight. The amount of X-gal produced was measured via absorbance at 600 nm (Fig. 6B). ![X-gal part registry](https://static.igem.org/mediawiki/2021/7/72/T--TUDelft--Part-registry-1.png "X-gal part registry") *Fig. 6 Comparison of X-gal production levels using the BBa\_I732017 (iGEM repository) and cRBS constructs at final concentration of 1 nM.* (A) Image of the PUREfrex2.0 reaction after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight. (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. Although the absorbance values of the cRBS construct indicated expression after 2 hours, it was 4.4 times lower compared to the control construct (Fig. 6). 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. Additionally, [modeling](https://2021.igem.org/Team%3ATUDelft/Model#Sensitivity) the aptazyme-regulated *lacZ* expression in the cell-free system suggested that lowering the DNA concentration has a positive impact on the target ligand concentration discernibility. However, the model also showed that lowering the DNA concentration slows down the expression. Given the low expression levels of our cRBS construct under the current DNA concentration, we considered that decreasing it even further could impose an additional limitation on the system’s speed and maximum expression level. Therefore, to keep AptaVita as a rapid and quantitative test, we designed a new genetic construct that could lead to a higher and faster expression, which we intended to have. Design of a new genetic construct We hypothesized that slow expression of cRBS construct was caused by the antisense strand remaining attached to the RBS even after the aptazyme self-cleavage. Therefore, we designed a new construct in which only part of the RBS is sequestered by the antisense strand (semi-cRBS, [BBa\_K3806016](http://parts.igem.org/Part%3ABBa_K3806016)). For a detailed explanation of this design, visit our [Design](https://2021.igem.org/Team%3ATUDelft/Design) page. We expressed both the cRBS and semi-cRBS constructs in the absence and presence of theophylline in PURExpress using CPRG as substrate. The product formation was determined by measuring the absorbance at 575 nm (Fig. 7). ![semi cRBS](https://static.igem.org/mediawiki/2021/6/6a/T--TUDelft--cRBS-semi-cRBS-abs.png "semi cRBS") *Fig. 7 Comparison of the colorimetric response between the cRBS and semi-cRBS designs added at a final concentration of 1 nM. 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. Expression profiles after 4 hours of expression indicated that the semi-cRBS was better expressed than our standard cRBS construct (Fig. 7). We also noticed that, as expected, the presence of theophylline results in reduced expression compared to the absence of theophylline in both of the designs. As an indication of both *(i)* **the dynamic range** represented by the absorbance difference between the smallest and highest ligand concentration (A0mM - A5mM), and *(ii)* **the response time** after 4 hours of expression, we calculated the area enclosed between the absorbance curves corresponding to 0 and 5 mM theophylline for both cRBS and semi-cRBS. For the cRBS design, the calculated area was 0.40 h x absorbance units (Au), whereas for the semi-cRBS the area was 1.03 h x Au, meaning a 2.57-fold increase. This indicates a larger dynamic range and faster response time when using the semi-cRBS construct. In addition, we determined the decrease in the expression level caused by the addition of 5 mM theophylline after 1.5 hours of expression as: ![Equation to calculate relative change ](https://static.igem.org/mediawiki/2021/e/e4/T--TUDelft--Relative_Change_LaTeX_Formula.PNG "Equation to calculate relative change") The semi-cRBS already shows a 33.3% decrease of expression after 1.5 hour, in contrast to the 6.1% decrease in the cRBS design, which confirms the improved response time of the system when using the semi-cRBS. To further validate our results, both designs were also expressed in triplicates using PUREfrex 2.0 and X-gal as substrate (Fig. 8). We again omitted the addition of theophylline as this gives the highest signal output. The product formation was measured at 600 nm. ![semi cRBS Xgal](https://static.igem.org/mediawiki/2021/6/61/T--TUDelft--X-gal-cRBS-semicRBS.png "semi cRBS Xgal") *Fig. 8 Comparison of X-gal production levels between the cRBS and semi-cRBS constructs in the absence of ligand. The genetic constructs were added at a final concentration of 1 nM* (A) Image of the PUREfrex2.0 reaction after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight (one of the replicates for each design is shown in this picture). (B) Absorbance measurement at 600 nm of the X-gal production. Data represents the mean ± SE (n = 3). The asterisk indicates a p-value < 0.05 in Student's t-test. As expected, the expression level was significantly higher for the semi-cRBS design, confirming that the results were consistent even if a different PURE cell-free system and substrate were used. Overall, we improved the performance of our biosensor by modifying our initial design enabling a more rapid and quantitative read-out. Ligand-responsive aptazyme cleavage in PURETesting the impact of theophylline addition on PURE Our final goal is to determine the ligand concentration by a colorimetric read-out measured via absorbance. Therefore, we tested the impact of theophylline on the colorimetric output of the system. Concerned that addition of theophylline in millimolar concentrations could have an impact on the expression system, we first performed a control experiment to analyze whether the decrease in expression observed after theophylline addition may result from interference of theophylline with the PURE expression system rather than from regulation by the ligand-responsive aptazyme. For this purpose, we again expressed the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_I732017%20) part, containing a *lacZ* gene without an aptazyme sequence, in PUREfrex2.0 using X-gal as substrate and increasing concentrations of theophylline. With this, we could assess the impact of theophylline on the PURE expression system independently of the aptazyme switch influence. ![Theophylline inhibition](https://static.igem.org/mediawiki/2021/6/6b/T--TUDelft--Inhibition.png "Theophylline inhibition") *Fig. 9 Image of the PUREfrex2.0 reactions after 2 hours of expression at 37 °C, and incubation with X-gal at room temperature overnight.* From left to right: (1) Negative control: no DNA template and 0 mM theophylline, (2) Negative control: no DNA template and 5 mM theophylline. Expression using BBa\_I732017 with (3) 0mM theophylline, (4) 1 mM theophylline, and (5) 5 mM theophylline. After two hours of expression at 37 °C with and without theophylline added (Fig. 13), we observed a decrease in the color intensity when increasing theophylline concentrations (Fig. 9, samples 3-5). This suggested that higher concentrations of theophylline have an inhibitory effect on the PURE expression system. To examine whether the ligand responsive expression was only due to theophylline inhibition or a combined effect of inhibition and aptazyme cleavage, we set up our next experiment. Cleavage characterization of the aptazyme-regulated genetic construct in PURE From the DRIVER experiments, we have proven that the theophylline aptazyme shows ligand-regulated cleavage activity in the T7 RNA polymerase reaction buffer (Fig. 1). However, the cleavage activity also needs to be validated in the PURExpress cell-free system. Since we saw expression inhibition by adding theophylline, we decided to decouple transcription and translation on our cell-free system reactions to address whether ligand-dependent cleavage of the atazyme also contributes to the observed decrease of expression. To do so, we first expressed our newly designed semi-cRBS construct in PURExpress for 1 hour (Fig. 2, step 1). The product of this first step was used as input for 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). From this assay, we expected to see a lower band intensity with decreasing concentration of theophylline. This means 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. 10 Scenario 1). However, if the ligand-dependent cleavage activity is not maintained, we expected to see the same band intensity regardless of theophylline concentracion (Fig. 10 Scenario 2). The region downstream the cleavage point was used as control for transcription, RT, PCR and loading in the agarose gel, as the band intensity should be similar between samples regardless of theophylline addition. ![RT qPCR](https://static.igem.org/mediawiki/2021/8/8b/T--TUDelft--RTqPCR.jpg "RT qPCR") *Fig. 10 Scheme of the 4 steps of the cleavage characterization procedure and potential scenarios.* Scenario 1 exemplifies ligand responsive self-cleavage of the aptazyme fused to *lacZ*, which is what we expected to take place in our system. Scenario 2 represents a ligand independent behavior of the system. The resulting agarose gel after the above mentioned procedure can be seen in Fig. 11. ImageJ was used to quantify the intensity (I) of the gel bands, and the uncleaved fraction was determined by: ![Equation to calculate uncleaved fraction](https://static.igem.org/mediawiki/2021/c/c2/T--TUDelft--Uncleaved_Fraction_LaTeX_Formula.PNG "Equation for uncleaved fraction") The computed uncleaved fractions were 36%, 52%, and 60% for 0, 1 and 5 mM theophylline, respectively. This means that the aptazyme preserves its theophylline-dependent cleavage activity in the PURExpress cell-free system (Fig. 10 Scenario 1). Additionally, the fusion of the *lacZ* gene to the aptazyme sequence does not hinder aptazyme self-cleavage. From these results we can conclude that even though we observed theophylline-caused inhibitory effects on the expression system (Fig. 9), the aptazyme presents a ligand-dependent self-cleavage activity in the desired working conditions (Fig. 16). Therefore, we hypothesize that the decrease in absorbance seen in Fig. 14 is caused by a combination of both theophylline inhibition, and aptazyme-regulated expression. Since the intensity of the control band was similar regardless of the theophylline concentration, we hypothesize that inhibition occurs at translation level. Fortunately, vitamin concentrations in human blood are in the nanomolar range, therefore we suspect that inhibition of the system in this range is less likely to occur [[3, 4]](#references). This could be confirmed by assessing the impact of the vitamins at human-like concentrations on the PURExpress cell-free system. ![gel RT qPCR](https://static.igem.org/mediawiki/2021/3/38/T--TUDelft--RTqPCR-gel.png "gel RT qPCR") *Fig. 11 Agarose gel analysis of the theophylline-dependent cleavage activity of the aptazyme in the semi-cRBS construct after expression in the semi-cRBS PURExpress.* (A) PCR product after amplification of the uncleaved fraction (displayed in the bottom of the gel). (B) PCR product after amplification of the control fragment. Lanes: (Ladder) dsDNA ladder. (1) Negative control without DNA template. (2) 0 mM theophylline (3) 1 mM theophylline. (4) 5 mM theophylline. Ligand-regulated expression on paper Next, we tested the ligand-regulated expression system 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 theophylline, and incubated at 37 °C for 24 hours. As expected, directly after rehydration, we did not see any color change of the paper disc for all of the tested conditions (Fig. 12, 0 h). After 24 hours of expression, however, we could see a slight color change from yellow to orange in the disc rehydrated with water and containing the semi-cRBS genetic construct (Fig. 12, 24 h, middle paper disc). ![paper based PURExpress](https://static.igem.org/mediawiki/2021/b/b3/T--TUDelft--Paper_FD.png "paper based PURExpress") *Fig. 12 Paper-based reactions using PURExpress after rehydration of the freeze-dried paper discs (0 h), and after 24 hours of expression at 37 °C (24 h).* The semi-cRBS construct was used at a concentration of 3 nM. From left to right: (1) without DNA template and 0 mM theophylline, (2) with DNA template and 0 mM theophylline, and (3) with DNA template and 5 mM theophylline. CPRG was used as a substrate to a final concentration of 0.6 mg/ml. We hypothesize that the moderate color change can be due to the loss of enzymatic activity during the freeze-drying process. This hypothesis is supported by a preliminary test that we performed with the cRBS construct omitting the freeze-drying step, which showed a strong color change for non-theophylline conditions (Fig. 13, 24 h, middle paper disc). Moreover, we did not observe a color change for theophylline conditions (right paper disc). This experiment shows the potential of AptaVita to be used as a paper-based platform for vitamin detection. Further adjustments in the freeze-drying process are needed to achieve the desired expression level to enable sterile and inexpensive transport of the biosensor. ![paper based PURExpress](https://static.igem.org/mediawiki/2021/0/08/T--TUDelft--Paper_NoFD.png "paper based PURExpress") *Fig. 13 Paper-based reactions using PURExpress after reaction assembly (0 h), and after 24 hours of expression at 37 °C (24 h).* The cRBS construct was used at a concentration of 3 nM as this experiment was performed before having built the semi-cRBS part. From left to right: (1) without DNA template and 0 mM theophylline, (2) with DNA template and 0 mM theophylline, and (3) with DNA template and 5 mM theophylline. CPRG was used as a substrate to a final concentration of 0.6 mg/ml. Achievements * We discovered that PURExpress is a compatible cell-free system to use with our desired substrate CPRG. * Comparison between our genetic construct (cRBS, [BBa\_K3806014](http://parts.igem.org/Part%3ABBa_K3806014)) and the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_I732017%20) part revealed low expression levels of our design. We created a new design (semi-cRBS, [BBa\_K3806016](http://parts.igem.org/Part%3ABBa_K3806016%20)) that showed an improved dynamic range compared to our previous design, enabling a more rapid and quantitative read-out. * From the expression of the [BBa\_I732017](http://parts.igem.org/Part%3ABBa_I732017%20) part in PURE with different theophylline concentrations we concluded that theophylline has inhibitory effects on the expression system. However, we showed that the aptazyme presents a ligand-dependent self-cleavage activity in the desired working conditions. This suggests that even though theophylline causes inhibition on the system, *lacZ* expression is also controlled by the theophylline-regulated aptazyme. * Expression of the genetic circuit on a paper support after freeze-drying resulted in a limited color change. On the other hand, expression without prior freeze-drying led to a larger color change. Adjustments in the freeze-drying procedure are required to enable inexpensive, sterile, and abiotic distribution of the envisioned test. Future prospectsIncorporation of novel vitamin biosensors Our theophylline-regulated aptazyme genetic construct provides an insight into the potential of AptaVita as a robust vitamin detection system. New vitamin-responsive aptazymes engineered through DRIVER should be incorporated in the genetic construct. To confirm the desired ligand-dependent colorimetric read-out, expression of the vitamin-responsive apatzyme circuit should be validated in a cell-free system on paper support. After optimization of the genetic circuit and paper preparation process, patient samples could be tested on the biosensor. Condition optimization to improve colorimetric read-out Freeze-drying procedure From the expression experiments using the theophylline construct, we saw that the impact of certain conditions on the aptazyme-regulated expression, for instance the freeze-drying procedure, could still be optimized for a better colorimetric read-out. As was observed in the paper expression results, only a slight color change was visible after freeze-drying of the paper. Adjustments on the freeze-drying procedure such as the freeze-drying time, or the addition of the substrate before freeze-drying or during rehydration, could potentially lead to an improved expression. Magnesium-dependent aptazyme self-cleavage The activity of most hammerhead ribozymes is Mg2+-dependent [[5]](#references). Preliminary results in our lab showed that the addition of extra rNTPs to the cell-free system reaction in order to chelate free Mg2+, has a positive effect on the stabilization of the aptazyme, resulting in less self-cleavage of the aptazyme in the absence of the ligand. Future experiments in which the Mg2+ concentration is varied could be used to gain knowledge on the optimum Mg2+ concentration to achieve the highest discernibility between vitamin concentrations. Paper and humidity Point-of-care tests need to be accurate in any environment in which they are used. This concern was also expressed by [local doctors and health organizations](https://2021.igem.org/Team%3ATUDelft/Human_Practices/iHP). The point-of-care tests should then be robust enough so they can be exposed to different storage and operating conditions and still give reproducible results. In Uganda, our target country for implementation, there are months where the humidity reaches 70%. Health organizations and experts told us that previous tests failed due to their bad performance under humid conditions. Therefore, we developed a detailed set-up for future experiments that recreates the humidity conditions in Uganda. Unfortunately, due to time restrictions, we could not fill in the required safety documents to comply with the iGEM safety and security policies. Therefore, we established a detailed experimental set-up for future purposes without performing the experiment. The aim of this experiment is to check: *(i)* how our test performs under humid conditions, and *(ii)* whether expression starts only after rehydration, and not during the storage time. To simulate the humidity conditions in Uganda, we would place our freeze-dried paper disks, containing the genetic construct, cell-free system, and CPRG substrate in the botanical garden of the Delft University of Technology. This garden has a room with an average temperature of 19-26 °C and 100% humidity. In the most positive outcome, the paper discs would not rehydrate within the storage time frame and would, after rehydration, show a similar expression as the control that is left in the lab (Fig. 14). This would give some first indications on whether our biosensor is able to be used under humid conditions. It is important to consider that this should be done after optimizing the freeze-drying conditions. A second optimization cycle might be needed to adjust the freeze-drying conditions for the paper-based biosensor to perform in a humid environment. ![Botanical garden experiment](https://static.igem.org/mediawiki/2021/d/d1/T--TUDelft--Botanical_garden_experiment.jpg "botanical garden experiment") *Fig. 14 Set-up for the humidity experiment.* The lyophilized papers contain the genetic construct, cell-free system, and CPRG substrate. The color of the paper disks is measured by a plate reader at every stage depicted in this figure. A microtiter plate will be stored for X days in the laboratory while the other plate will be stored for the same amount of time in the botanical garden in humid conditions. Both microtiter plates are measured again to check if rehydration did already (partly) occur during the storage time. Afterwards, both microtiter plates are rehydrated to check if they give the same results or if storage under humid conditions influences the paper disks. Paper-based microfluidic chip To improve the user-friendliness of the biosensor, we are interested in detecting multiple vitamins by a single rehydration step of the paper disks. For this purpose, we looked into using a microfluidic approach on a laser-printed paper, which has been reported to be a successful strategy by Ghosh *et al.* [[6]](#references). We performed some pilot experiments to test whether the ink could create a hydrophobic layer by printing a pattern on chromatography paper consisting of two wells connected through a channel. Paper-based microfluidic chip pilot experiments By heating-up the paper, we expected the ink to melt and impregnate the paper to create a hydrophobic channel that is able to create a microfluidic flow for hydrophilic substances. To easily follow the liquid trajectory, we tested our paper design with blue-colored milli-Q water. We observed that the ink creates a hydrophobic layer that conserves the water in the well and channel (Supplementary Fig. 8). Unfortunately, we did not see the water flow through the channels as we expected it to do. In order to fix this, future experiments should then focus on testing different channel thicknesses, channel length, optimal shape, and patterns and test the fluidics and flow speed of more patient sample related fluids and conditions to allow for a single rehydration step in our biosensor ![microfluidics front](https://static.igem.org/mediawiki/2021/c/c6/T--TUDelft--microfluidics_front.jpeg "microfluidics front") ![microfluidics back](https://static.igem.org/mediawiki/2021/5/5e/T--TUDelft--microfluidics_back.jpeg "microfluidics back") *Supplementary Fig. 8 The hydrophobic capacities of our printed pattern tested with blue-colored water.* The patterns are laser printed on chromatography paper. The printer setting used is 600 dots per inch (DPI), reprinted 3 times.

## Hardware

To make our test available at the point of care, we developed a frugal read-out device that measures absorbance over time to quantify the color-based outcome of the AptaVita test. For more information on the read-out device, visit our [Hardware](https://2021.igem.org/Team%3ATUDelft/Hardware) page

### Blocking ambient light

For the measurements, we needed to ensure that the test box is opaque to minimize the ambient light coming through the test box and reaching light sensors. To this end, the test box and the inside of the embodiment were spray painted with black matte spray. We measured the ambient light that reached each sensor for four different conditions (Tab. 2). The results are shown in Fig. 16. For each sensor, no ambient light was measured in the fourth condition, which is the same as the test condition. Hereby, we show that the ambient light is sufficiently blocked.

*Tab. 2 Overview of the test conditions of the ambient light blocking experiment.*

|  | Closed embodiment | Closed test box | Test cassette in test box |
| --- | --- | --- | --- |
| Condition 1 | - | - | - |
| Condition 2 | - | - | + |
| Condition 3 | - | + | + |
| Condition 4 | + | + | + |

![Different light conditions](https://static.igem.org/mediawiki/2021/7/7e/T--TUDelft--Hardware_Different_Light_Conditions.svg "Different light conditions")

*Fig. 16 Percentage of transmitted light relative to the light intensity that was measured with an opened device.* The light intensity was measured in four different conditions, which are summarized in Tab. 2. For each sensor, the percentage of transmitted light was calculated by normalizing with respect to condition 1. The measurements of a sensor were averaged over four minutes to account for fluctuations in the incoming light. The standard error of the mean was lower than 0.12% for all conditions and sensors.

### Proof-of-concept for concentration measurements

Our final goal is to measure the CPR concentration over time during a test. The measured concentration curve can be linked to the vitamin concentration of a sample. In this section, we demonstrate the ability of our device to measure colorimetric differences between CPR concentrations at the pH of blood plasma (pH = 7.4) [[7]](#references).

First, we measured the absorbance spectrum of CPR on a paper disc in a plate reader to validate that CPR absorbs light in the range of wavelengths emitted by the LEDs (550 - 600 nm) of our read-out device. This was the case, as we observed an absorbance peak at 579 nm (Fig. 17).

To measure the absorbance at a range of CPR concentrations, we linearly diluted the CPR to 0.25 mM, 0.5 mM, 0.75mM, 1mM, and 1.25 mM (see Fig. 17). For each concentration, the absorbance was measured in duplicate in the plate reader, and triplicate in the read-out device. As a blank measurement, we used a paper disc that was hydrated with the same buffer as used for the dilution. In the plate reader, we observe a linear relation with an R2 of 0.97 (Fig. 18a). This shows that the Beer-Lambert law is valid for this range of CPR concentrations. In the read-out device, we also observed a linear relation between the absorbance and the CPR concentration with an R2 of 0.95 (Fig. 18b). This demonstrates that the device is capable of distinguishing different CPR concentrations through absorbance measurements.

![CPR spectrum](https://static.igem.org/mediawiki/2021/4/4c/T--TUDelft--CPR_Absorbance_Spectrum.svg "CPR spectrum")

*Fig. 17 Absorbance spectrum of CPR on a paper disc measured with a plate reader.* The disc was hydrated with 5.5 µL of 0.25 mM CPR. The CPR was diluted with Phosphate-Buffered Saline (PBS). A disc with 5.5 µL PBS was used as a blank measurement.

![CPR absorbance curves hardware](https://static.igem.org/mediawiki/2021/8/85/T--TUDelft--CPR_Absorbance_Curves_Hardware_Plate.svg "CPR absorbance curves hardware")

*Fig. 18 Absorbance against CPR concentration on paper discs.* The CPR was diluted with PBS. Each paper disc was hydrated with 5.5 µL of the CPR dilution. A linear curve was fitter for both measurements. (a) Absorbance measurements by a plate reader (y = 0.61 x + 0.16, R2 = 0.97). (b) Absorbance measurements by the AptaVita read-out device (y = 0.80 x + 0.137, R2 = 0.95).

### Achievements

1. We show that our device is able to reduce the ambient light to a level which cannot be measured by the read-out device.
2. We demonstrate that the hardware is capable of distinguishing different CPR concentrations through absorbances measurements.

### Future Prospects

* By improving the positioning of the LEDs and reorganizing the cables, the amount of light that reaches the sensor can be increased, so that a larger part of the range of the sensor is used, thereby increasing the precision and sensitivity of the measurements.
* Although we showed that our device is capable of performing measurements on the same disc over a time period, we have not tested if the device can measure a change of CPR concentration over time. This would be the next step in testing our read-out device. In the end, the measured temporal evolution of the CPR concentration could be compared to reference curves that correspond to known vitamin concentrations. The vitamin concentration of the test sample could be inferred from the comparison.

## 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. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kalé, L., & Schulten, K. (2005). Scalable molecular dynamics with NAMD. Journal of computational chemistry, 26(16), 1781–1802. <https://doi.org/10.1002/jcc.20289>
3. Center for Disease Control and Prevention, World Health Organization, Nutrition International, UNICEF. *Micronutrient survey manual.* Geneva: World Health Organization; 2020. Licence: CC BY-NCSA 3.0 IGO. <https://www.who.int/publications/i/item/9789240012691>
4. Albaugh, V.L., Williams, D.B., Aher, C.V., Spann, M.D., & English, W.J. (2021). Prevalence of thiamine deficiency is significant in patients undergoing primary bariatric surgery. *Surgery for Obesity and Related Diseases*, **17**(4), 653-658. <https://doi.org/10.1016/j.soard.2020.11.032>
5. Naghdi, M. R., Boutet, E., Mucha, C., Ouellet, J., & Perreault, J. (2020). Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium. *Non-coding RNA, 6*(1)., 14. <https://doi.org/10.3390/ncrna6010014>
6. Ghosh, R., Gopalakrishnan, S., Savitha, R., Renganathan, T., & Pushpavanam, S. (2019). Fabrication of laser printed microfluidic paper-based analytical devices (LP-µPADs) for point-of-care applications. *Scientific reports, 9*(1), 1-11.
7. Aoi, W., & Marunaka, Y. (2014). Importance of pH homeostasis in metabolic health and diseases: crucial role of membrane proton transport. *BioMed research international*, 2014, 598986. <https://doi.org/10.1155/2014/598986>

## A big thank you to our sponsors!

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