iGEM 2021 — Team Thessaloniki

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# Team:Thessaloniki/Notebook

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* ## PROJECT
  + [| Description](https://2021.igem.org/Team%3AThessaloniki/Description)
  + [| Entrepreneurship](https://2021.igem.org/Team%3AThessaloniki/Entrepreneurship)
  + [| Model](https://2021.igem.org/Team%3AThessaloniki/Model)
  + [| Results](https://2021.igem.org/Team%3AThessaloniki/Results)
  + [| Software](https://2021.igem.org/Team%3AThessaloniki/Software)
  + [| Medals](https://2021.igem.org/Team%3AThessaloniki/Medals)
  + [| Hardware](https://2021.igem.org/Team%3AThessaloniki/Hardware)
  + [| Resources Table](https://2021.igem.org/Team%3AThessaloniki/Resources_Table)
  + [| Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)
  + [| Wiki Map](https://2021.igem.org/Team%3AThessaloniki/Wiki_Map)
  + [| Implementation](https://2021.igem.org/Team%3AThessaloniki/Implementation)
  + [| Judging Form](https://igem.org/2021_Judging_Form?id=3727)
  + [| Proof of Concept](https://2021.igem.org/Team%3AThessaloniki/Proof_Of_Concept)
  + [| Our Inspiration](https://2021.igem.org/Team%3AThessaloniki/Inspiration)
  + [| Contribution](https://2021.igem.org/Team%3AThessaloniki/Contribution)
* ## OUTREACH
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Human_Practices)
  + [| Collaborations](https://2021.igem.org/Team%3AThessaloniki/Collaborations)
  + [| Partnership](https://2021.igem.org/Team%3AThessaloniki/Partnership)
  + [| Communication](https://2021.igem.org/Team%3AThessaloniki/Communication)
  + [| Education](https://2021.igem.org/Team%3AThessaloniki/Education)
  + [| Promotion](https://2021.igem.org/Team%3AThessaloniki/Communication/Promotion)
* ## TEAM
  + [| PI](https://2021.igem.org/Team%3AThessaloniki/Team#PI)
  + [| Members](https://2021.igem.org/Team%3AThessaloniki/Team#Members)
  + [| Mentors](https://2021.igem.org/Team%3AThessaloniki/Team#mentors)
  + [| Advisors](https://2021.igem.org/Team%3AThessaloniki/Team#advisors)
  + [| Attributions](https://2021.igem.org/Team%3AThessaloniki/Attributions)
* ## NOTEBOOK
  + [| Team](https://2021.igem.org/Team%3AThessaloniki/Notebook)
  + [| Photo Album](https://2021.igem.org/Team%3AThessaloniki/Notebook/Photos)
  + [| Wet Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
  + [| Dry Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Dry)
  + [| FR](https://2021.igem.org/Team%3AThessaloniki/Notebook/FR)
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Notebook/Human)
* ## LAB
  + [| Parts](https://2021.igem.org/Team%3AThessaloniki/Parts)
  + [| Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
  + [| Safety](https://2021.igem.org/Team%3AThessaloniki/Safety)
* ## SPONSORS
  + [| Sponsors](https://2021.igem.org/Team%3AThessaloniki/Sponsors)
  + [| Become a Sponsor](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Become)
  + [| Special Thanks](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Thanks)

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
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igemthessaloniki@gmail.com

## Team's Notebook

[![](https://static.igem.org/mediawiki/2021/f/fa/T--Thessaloniki--scroll_down.png)](#up)

### More Notebooks:

[* ##### Wet Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
[* ##### Dry Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Dry)

[* ##### Human Practices](https://2021.igem.org/Team%3AThessaloniki/Notebook/Human)
[* ##### Fundraising](https://2021.igem.org/Team%3AThessaloniki/Notebook/FR)

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* | Greece, Thessaloniki
* | Aristotle University
* | School of Sciences
* | Department of Biology

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)
* | igemthessaloniki@gmail.com

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

# Team:Thessaloniki/Engineering

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×

* ## PROJECT
  + [| Description](https://2021.igem.org/Team%3AThessaloniki/Description)
  + [| Entrepreneurship](https://2021.igem.org/Team%3AThessaloniki/Entrepreneurship)
  + [| Model](https://2021.igem.org/Team%3AThessaloniki/Model)
  + [| Results](https://2021.igem.org/Team%3AThessaloniki/Results)
  + [| Software](https://2021.igem.org/Team%3AThessaloniki/Software)
  + [| Medals](https://2021.igem.org/Team%3AThessaloniki/Medals)
  + [| Hardware](https://2021.igem.org/Team%3AThessaloniki/Hardware)
  + [| Resources Table](https://2021.igem.org/Team%3AThessaloniki/Resources_Table)
  + [| Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)
  + [| Wiki Map](https://2021.igem.org/Team%3AThessaloniki/Wiki_Map)
  + [| Implementation](https://2021.igem.org/Team%3AThessaloniki/Implementation)
  + [| Judging Form](https://igem.org/2021_Judging_Form?id=3727)
  + [| Proof of Concept](https://2021.igem.org/Team%3AThessaloniki/Proof_Of_Concept)
  + [| Our Inspiration](https://2021.igem.org/Team%3AThessaloniki/Inspiration)
  + [| Contribution](https://2021.igem.org/Team%3AThessaloniki/Contribution)
* ## OUTREACH
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Human_Practices)
  + [| Collaborations](https://2021.igem.org/Team%3AThessaloniki/Collaborations)
  + [| Partnership](https://2021.igem.org/Team%3AThessaloniki/Partnership)
  + [| Communication](https://2021.igem.org/Team%3AThessaloniki/Communication)
  + [| Education](https://2021.igem.org/Team%3AThessaloniki/Education)
  + [| Promotion](https://2021.igem.org/Team%3AThessaloniki/Communication/Promotion)
* ## TEAM
  + [| PI](https://2021.igem.org/Team%3AThessaloniki/Team#PI)
  + [| Members](https://2021.igem.org/Team%3AThessaloniki/Team#Members)
  + [| Mentors](https://2021.igem.org/Team%3AThessaloniki/Team#mentors)
  + [| Advisors](https://2021.igem.org/Team%3AThessaloniki/Team#advisors)
  + [| Attributions](https://2021.igem.org/Team%3AThessaloniki/Attributions)
* ## NOTEBOOK
  + [| Team](https://2021.igem.org/Team%3AThessaloniki/Notebook)
  + [| Photo Album](https://2021.igem.org/Team%3AThessaloniki/Notebook/Photos)
  + [| Wet Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
  + [| Dry Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Dry)
  + [| FR](https://2021.igem.org/Team%3AThessaloniki/Notebook/FR)
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Notebook/Human)
* ## LAB
  + [| Parts](https://2021.igem.org/Team%3AThessaloniki/Parts)
  + [| Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
  + [| Safety](https://2021.igem.org/Team%3AThessaloniki/Safety)
* ## SPONSORS
  + [| Sponsors](https://2021.igem.org/Team%3AThessaloniki/Sponsors)
  + [| Become a Sponsor](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Become)
  + [| Special Thanks](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Thanks)

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)

igemthessaloniki@gmail.com

[* Overview](#over)
[* 1st Direction](#first)
[* 2nd Direction](#second)
[* 3rd Direction](#third)
[* Summary](#sum)
[* References](#ref)

## Engineering Success

[![](https://static.igem.org/mediawiki/2021/f/fa/T--Thessaloniki--scroll_down.png)](#up)

## Overview

For our engineering success, our main goal was the design, evaluation, and improvement of our biological system. Our designed system was based upon the technology of synthetic riboregulators called toehold switches. These molecules are designed to detect some specific miRNAs that are present during Pancreatic Ductal Adenocarcinoma, the most common type of pancreatic cancer. The process of engineering includes the detailed design of those synthetic riboregulators that are built and tested in the laboratory. Also, it includes the experiments for fluorescence measurement for their evaluation and improvement. The improvements concerned the replacement of the RBS for the second generation sequences and also, the selection of different reporter genes. In our case, the different reporter genes are different fluorescent proteins. Moreover, since it is crucial to store this sensitive synthetic system, we cloned our parts. Finally, we designed and tested an isothermal amplification protocol, the so-called EXPAR, to increase the sensitivity of our system. So, our engineering success is built upon these three main directions.

## 1st Direction

The main goal of our project is the early detection of adenocarcinoma in the pancreas. This certain type of cancer was chosen because its diagnosis is made in the last stages (III and IV) [1], and thus it is considered one of the most deadly forms of cancer [2]. Our goal is to diagnose PDAC in its early stages by detecting three upregulated miRs, miRNA 143-3p, 30e-5p, and 1246 in urine, which were selected from literature, and their importance was confirmed through extensive bioinformatics analysis. The detection was performed using synthetic RNA sequences called Toehold Switches.

### | Engineering cycle 1

In the first engineering cycle, the RBS part in synthetic toehold switches sequences was modified. The first generation parts contained the RBS sequence used by Green et al. The second generation contained a different RBS, the RBS-DHFR, suggested by the manufacturer of the cell-free system we used. This modification was made to check whether the fluorescence levels measured were increased.

**~Design**

For our project, we chose the trigger RNAs hsa-miR-143-3e, hsa-miR-30e-5p, and hsa-miR-1246. Our synthetic sequences comprise of the complementary to the miRNA sequences, followed by the RBS, the AUG codon, and the Linker, which are mentioned in the article of Green [3]. The reporter gene selected was eGFP, which has a maximum absorption spectrum at 488 nm and an emission spectrum at 507 nm. The sequences were designed to avoid termination codons and restriction sites for the restriction enzymes we used.

From these sequences, 3 were selected. More specifically, we selected one sequence per miRNA. They showed the best and the most promising results since the thermodynamic parameter of Gibbs free energy from the RBS part to the end of the Linker was close to zero. This energy was calculated by ViennaRNA and NUPACK, software designed for studying nucleic acid interactions. This parameter is a thermodynamic property that is considered to have the highest correlation with the functionality of the toehold switch-trigger RNA system [3].

You can check our Model page for a more detailed description:

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

**~Build**

In accordance with the design, we ordered specific sequences in the form of double-stranded, DNA (g –Blocks) from IDT.

Specifically, the following parts were used:

* ~[BBa\_K3727017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727017)
* ~[BBa\_K3727026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727026)
* ~[BBa\_K3727029](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727029)

For these experiments, we used both a negative and a positive control sample. As a negative control, we used all the reagents from the cell-free system (NEB’s PURExpress® *In Vitro* Protein Synthesis Kit #E8600), while as a positive control we used the same volume of cell-free system reagents and the DHFR plasmid.

**~Test**

In order to test the functionality of Toehold switches designed, we executed a series of experiments using a cell-free system (NEB - PURExpress® *In Vitro* Protein Synthesis Kit #E8600). These experimental procedures aimed to prove the stability of our Toeholds; that is the unfolding of the hairpin as well as the production of eGFP protein, only in the presence of the trigger RNA. In our case, the trigger RNA is the miRNA. For this purpose, we used black 96-well-plates (Greiner #655077) and in each well, we added:

* **1.** The toehold switches, BBa\_K3727017, BBa\_K3727026, BBa\_K3727029 respectively, with the reagents of NEB - PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600), in three separate wells. The toehold switches’ quantity was 30ng per 25 ul reaction.
* **2.** The toehold switches, as described above, with the complementary miRNA mimics (Thermofisher - mirVana® miRNA mimic #4464066). The miRNAs’ concentration was 10nM, in three separate wells.
* **3.** The reagents of the NEB’s cell-free system PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600), as a negative control.
* **4.** The reagents of the cell-free system NEB - PURExpress® and the DHFR plasmid as a positive control, in case of electrophoresis. The DHFR control template is supplied at 125 ng/μl. So, we used 2 ul for the positive control reaction.

The reagents used in the experiments are in Table 1:

| Reagents | Volume |
| --- | --- |
| Solution A | 10 ul |
| Solution B | 7.5 ul |
| RNAse inhibitor | 0.5 ul |
| Template DNA | 30 ng |
| Nuclease free water | to 25 ul |

Table 1. Reagents used in the fluorescence measurements.

For the measurement of the fluorescence, we used the device “Victory-X 3000” from Perkin Elmer company.

**~Learn**

The measurements conducted adhere to the excitation/emission protocol of 485/535 nm.

From the results of these experiments, we concluded that in order to increase the ON/OFF ratio, it is essential to increase the fluorescence that is detected. The ON/OF ratio parameter is crucial for the quantification and hence, for the formation of the standard curve of fluorescence.

The improvement of the system can be achieved through a different design of toehold switches. To achieve higher expression of the reporter gene of eGFP, we tried incorporating a different RBS. Since we utilized NEB’s cell-free system during fluorescence measurements, we chose the DHFR plasmid RBS. This way we achieved a higher affinity between the RBS and the ribosome and improved the expression of the reporter gene.

![](https://static.igem.org/mediawiki/2021/f/f7/T--Thessaloniki--first_engineering_cycle_on_off.png)

Figure 1. This graph presents the fluorescence produced by Toehold switches in the presence of the complementary miR mimic (Thermofisher - mirVana® miRNA mimic #4464066) in comparison with the fluorescence produced by the Toehold switch in the absence of miRNA (Negative Control).

### | Engineering cycle 2

In this cycle, the second generation of toehold switches was designed and tested experimentally. From the results obtained, it seemed necessary to optimize the conditions of the experiments and use another fluorescent protein to avoid the signal received due to background fluorescence.

**~Design**

The second generation of toehold switches was designed to be more compatible with the ribosomes of the cell-free system. For this reason, the sequences of the second generation of toeholds followed the same structure as the previous ones with one exception. The RBS used for this generation was the RBS from the DHFR plasmid, which is supplied with the cell-free system we used.

**~Build**

In accordance with the design, we ordered specific sequences in the form of double-stranded DNA (g –Blocks) from IDT.

Specifically, the following parts were used:

* ~[BBa\_K3727016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727016)
* ~[BBa\_K3727027](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727027)
* ~[BBa\_K3727028](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3727028)

**~Test**

In order to test the functionality of Toehold switches designed, we executed a series of experiments using a cell-free system NEB’s PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600). These experimental procedures aimed to prove the stability of our Toeholds; that is the unfolding of the hairpin as well as the production of eGFP protein, only in the presence of the trigger RNA. In our case, the trigger RNA is the miRNA. For this purpose, we used black, 96-well-plates (Greiner #655077) and in each well, we added:

* **1.** The toehold switches BBa\_K3727016, BBa\_K3727027, BBa\_K3727028 respectively, with the reagents of NEB’s PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600), in three separate wells. The toehold switches’ quantity was 30ng per 25ul reaction.
* **2.**The toehold switches, as described above, with the complementary miRNA mimics (Thermofisher - mirVana® miRNA mimic #4464066). The miRNAs’ concentration was 10nM, in three separate wells.
* **3.** The reagents of the NEB’s cell-free system PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600), as a negative control.
* **4.** The reagents of the NEB’s cell-free system PURExpress® and the DHFR plasmid as a positive control, in case of electrophoresis. The DHFR control template is supplied at 125 ng/μl. So, we used 2 ul for the positive control reaction.

The reagents used in the experiments are in Table 2:

| Reagents | Volume |
| --- | --- |
| Solution A | 10 ul |
| Solution B | 7.5 ul |
| RNAse inhibitor | 0.5 ul |
| Template DNA | 30 ng |
| Nuclease free water | to 25 ul |

Table 2. Reagents used in the fluorescence measurements.

For the measurement of the fluorescence, we used the device “Victory-X 3000” from Perkin Elmer company.

**~Learn**

The measurements conducted adhere to the excitation/emission protocol of 485/535 nm.

![](https://static.igem.org/mediawiki/2021/e/e1/T--Thessaloniki--second_engineering_cycle_on_off.png)

Figure 2. This graph represents the results of fluorescence experiments of Toehold switches BBa\_K3727016 with miRNA 143-3p, BBa\_K3727027 with miRNA 30e-5p, and BBa\_K3727028 with miRNA 1246. The control bar shows the fluorescence of the well, which includes only the reagents of the “PURExpress *In vitro* Protein Synthesis kit” (#E6800) as a negative control. The second bar shows the fluorescence produced by Toehold switches in the presence of the complementary miR mimic (ON) (Thermofisher - mirVana® miRNA mimic #4464066) and the third one, the fluorescence produced by the Toehold switch in the absence of miRNA (OFF).

**~Improved results using the protocol at 488/507 nm**

According to the literature, the eGFP excitation spectrum has a peak at 488 nm, while its emission spectrum has a peak at 507 nm. To achieve a more accurate measurement, we used the device of Promega “GloMax-Multi Detection System” which has a special protocol and filter for the measurement of fluorescence of this protein. The spectrum of emission and excitation of eGFP is demonstrated in the next diagram.
~Data was taken from FPBase~

![](https://static.igem.org/mediawiki/2021/a/ab/T--Thessaloniki--eGFP.png)

Figure 3.The emission and absorption spectrum of eGFP

![](https://static.igem.org/mediawiki/2021/f/f6/T--Thessaloniki--improvement_on_off.png)

Figure 4. This graph represents the results of fluorescence experiments of Toehold switches BBa\_K3727016 with miRNA 143-3p, BBa\_K3727027 with miRNA 30e-5p, and BBa\_K3727028 with miRNA 1246. The control bar shows the fluorescence of the well, which includes only the reagents of the “PURExpress *In vitro* Protein Synthesis kit” (#E6800) as a control. The second bar shows the fluorescence produced by the Toehold switch in the presence of the complementary miR mimic (ON) (Thermofisher - mirVana® miRNA mimic #4464066) and the third one, the fluorescence produced by the Toehold switch in absence of miRNA (OFF).

From the results of this protocol, we concluded that the background fluorescence of the cell-free system is high. That means this specific protocol is not capable of detecting the low fluorescence produced only by the eGFP. Since the detection of small amounts of fluorescence is not achieved, then the detection of small amounts of miRNA can not be detected. To improve the ON/OFF ratio, it was important to find an appropriate protocol in which the cell-free system did not produce fluorescence. For that reason, an experiment to measure the background fluorescence in a different protocol was performed. The protocol chosen was designed for the fluorescent protein EBFP. The spectrum of emission and excitation of EBFP is shown in the next diagram.~The data used was taken from FPBase.~

![](https://static.igem.org/mediawiki/2021/c/c4/T--Thessaloniki--EBFP.png)

Figure 5.The emission and absorption spectrum of eBFP.

![](https://static.igem.org/mediawiki/2021/8/87/T--Thessaloniki--EGFP_VS_EBFP.png)

Figure 6. Background fluorescence of NEB’s cell-free system(#E6800) for 2 different protocols : 1) at 488/507 nm, 2) at 380/420 nm.

The excitation/emission protocol used for the measurement of the background fluorescence of the cell-free system was 488/507 nm and 380/420 nm. These specific nanometer wavelengths correspond to the peaks of eGFP and EBFP spectra, as shown in Figures 3 and 5. From the results of this experiment, we can conclude that the detected fluorescence for the EBFP protocol is decreased in comparison with the fluorescence detected from the eGFP protocol.

Moreover, EBFP has several advantages compared to eGFP. The overlap of the two EBFP spectra is smaller compared to the overlap of eGFP’s spectra. This can be validated by calculating the ratio of the overlap area to the whole area of the spectra for each protein. Specifically for the protein eGFP, this ratio is calculated at 0.139 and for the protein EBFP, this ratio can be calculated at 0.075. The decrease in that ratio means that the interference effect is minimized and the process of fluorescence detection is more accurate. So, the use of this specific protein as a reporter gene is likely to improve our system’s functionality for detecting small amounts of miRNAs and in general to increase the ON/OFF ratio.

## 2nd Direction

After designing and ordering our parts we decided to clone them into an appropriate vector to be able to amplify and store them while also avoiding possible mutations so that they could be intact for future use.

Our attempted cloning procedures include 2 separate cloning methods; BioBrick Assembly and PCR Cloning. Our efforts can be categorized into 2 engineering cycles which are analyzed below. In the first one we utilized the BioBrick Assembly method and in the second one the PCR Cloning procedure.

### | Engineering cycle 1

**~Design**

We designed our parts by combining the toehold switch sequence and the downstream gene eGFP while also adding the BioBrick prefix and suffix so that they could be cloned. We also added two leader sequences upstream of the BioBrick prefix and downstream of the BioBrick suffix, so that the restriction enzymes would be able to bind to the DNA sequence and cut at the appropriate site. These sequences would be cut off after the digestion of the parts and are not inserted into the vector - hence, they are not considered a section of the part.

The main layout of our components is the following:

**Leader Sequence --> BioBrick Prefix --> T7 Promoter --> Toehold Switch --> eGFP --> T7 Terminator --> Biobrick suffix --> Leader Sequence**

We designed our experiment protocols on the Benchling platform and simulated all the procedures with the Assembly Wizard tool.

![](https://static.igem.org/mediawiki/2021/f/f2/T--Thessaloniki--pSB1C3-T-30e-5p-1.png)

Figure 7: This is the plasmid map of one of the Toehold Switches we cloned, and more specifically T-30e-5p-1, created by Assembly Wizard. For this simulation we indicated the restriction enzymes we decided to use, that is EcoRI and PstI and the software automatically created the map and designated possible mistakes. We got no error messages, which means that our part can be cloned in the desired vector. Here we provide only this particular example of our simulations because all the parts have the same layout.

We also simulated the restriction digestions using the virtual digestion tool to ensure that the restriction sites were appropriate for this cloning method.

![](https://static.igem.org/mediawiki/2021/9/91/T--Thessaloniki--digetstion_ruler.png)

Figure 8: In this figure the sizes of the linearized vector and the digested g-Block are shown. This tool helped us in understanding the sizes and the position of the bands we expected to see on the gel after electrophoresis and as a result the appropriate time to run the gel. The sample on the left is the digested g-Block and the bands at 75 bp correspond to the edges of the g-Block. These fragments are very small -approximately 15 bp each- and can not be visualized through electrophoresis.

The results of our simulations proved that our parts are compatible with the BioBrick Assembly method as specified by the iGEM Competition.

**~Build**

We used 2 separate resources to decide on our protocol, [4] and [5]. The cloning pipeline we followed consisted of a series of experimental techniques which are shown in chronological order:

* **1.** g-Block Resuspension
* **2.** g-Block Digestion
* **3.** Vector Digestion
* **4.** Ligation of insert and vector
* **5.** Transformation in competent cells
* **6.** Inoculation of liquid bacterial cultures
* **7.** Plasmid DNA isolation with minipreps
* **8.** Diagnostic Restriction Digestion
* **9.** Sanger Sequencing

In the first step, we also need to mention that we aliquoted the resuspended g-Blocks to avoid contamination and many unwanted freeze-thaw cycles.

You can find these protocols in our Experiments Page in the section "Cloning":

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

**~Test**

In this part of our engineering cycle, we tested our protocols as defined above, and we tried different conditions to achieve the best possible results. Unfortunately, we were not able to obtain any colonies out of our transformation efforts.

As mentioned above the first steps of our pipeline concern the digestion of the g-blocks and the vector. We conducted these experiments as described in the corresponding protocols and ran electrophoresis of the samples. We got the same image as expected from our simulations, which means that there were no mistakes in these procedures that could be responsible for our results.

Initially, we used a ratio of 1:3 insert:vector in our ligation procedure, but did not get promising results, so after troubleshooting and consulting both the supervisors in our lab, our mentors, and our partners from iGEM Patras, we changed the ratio to 1:5 and even later on, to 1:7. Also, we used a combination of the above, with different ligation conditions. At first, we used an incubation at room temperature for a short period of time - 15 to 20 minutes - and then heat inactivation at 80°C or we incubated the ligated product at 16°C overnight. Regrettably, all of the above did not bear any encouraging results.

In every transformation, we used one plate for a positive control to see whether the problem was the competent cells we were using but still, this was not the issue. Our positive control had a lot of colonies whereas our samples were empty. So, we gathered that our competent cells were not ineffective, as to our ligation which was indeed problematic.

After many unsuccessful efforts to clone our parts, we decided to test if we can digest the vector only and re-ligate the linearized part and its insert. We used the same protocols as mentioned above, but still, we got no colonies. This way we concluded that there was no problem with the design of our parts, but with the ligation protocol itself.

Furthermore, during these experiments, we found out that the aliquots of the resuspended g-Blocks had different concentrations and in many of them it was very low. This could also be a problem that held back our cloning.

**~Learn**

Although we got no results, through troubleshooting, we came to a conclusion of what was wrong and found possible solutions. Unfortunately, due to the pressuring timeline of the competition, we were not able to test all the suggested alternatives from the troubleshooting of the first engineering cycle so we decided to proceed with the PCR cloning method to see whether we would receive better results.

Below, we are citing the solutions we came up with and the factors we believe they would affect.

| Troubleshooting | Possible explanations |
| --- | --- |
| Different T4 ligase | Increase the ligation efficiency |
| Extended ligation time | Increase the possibility of a successful ligated product |
| Different growth medium | Increase the growth of transformed cells during the outgrowth step of transformation. |
| Reorder the parts and repeat the resuspension | Achieve a better concentration of our toeholds |

Table 3: Troubleshooting for cloning procedure

### | Engineering Cycle 2

**~Design**

We used the same parts but this time we used PCR to amplify our sequences. For the design of our PCR, we used the Tm Calculator from NEB to ensure the best annealing temperature with regard to our primers’ nucleotide number, Tm, and GC content.

We also used the Benchling platform to cross-check the primers’ Tm which showed a slight difference so we tried both of the suggested annealing temperatures.

**~Build**

For this procedure, we used NEB's “PCR Cloning Kit” and we organized our experiments based on the protocol supplied by the manufacturer. The pipeline of these experiments is the following:

* **1.** Primer preparation
* **2.** Amplification of g-Blocks with PCR
* **3.** Gel extraction
* **4.** Ligation of insert and vector
* **5.** Transformation in competent cells
* **6.** Inoculation of liquid bacterial cultures
* **7.** Plasmid DNA isolation with minipreps
* **8.** Diagnostic Restriction Digestion
* **9.** Electrophoresis
* **10.** Sanger Sequencing

You can also find these protocols on our Experiments page:

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

For the amplification of the parts with PCR, we used the “Q5-High Fidelity Master Mix” from NEB and we tried different settings, based on the corresponding protocol.

**~Test**

The bottleneck for this cycle was to achieve a satisfying amplification of the toeholds with PCR so we tried different approaches to succeed it. In this part of the engineering cycle, we used different temperature conditions for each PCR step. Specifically, we used both of the suggested Tms from Benchling and NEB Tm Calculator and we also changed the time of every step after troubleshooting and many consultations. As a way to find the best conditions, we changed the PCR cycles from 30 to 35 to receive a better final concentration.

After determining the appropriate annealing temperature, we experimented with different concentrations of the components of the reaction. Since our g-Blocks are resuspended in TE buffer, which contains EDTA that chelates Mg2+, we tried different concentrations of Mg2+. The concentration spectrum was 2-4 mM. Higher Mg2+ concentration increased the yield but also increased the byproducts of the reaction.

![](https://static.igem.org/mediawiki/2021/5/51/T--Thessaloniki--PCR.jpg)

Figure 9: This is a photo of three PCR samples. For the first one, we initially added half the quantity of DNA of the other two samples and the higher Mg2+ quantity. The other two samples had the same quantity of DNA, but the first one had extra Mg2+. This experiment confirms the above, that the Mg2+ increases both the efficiency of the amplification but also, reduces the purity of the product.

After we succeeded to achieve a satisfying PCR result we were able to continue the procedure with the next steps as indicated in the Build section.

**~Learn**

In the end, we were finally able to clone 15 out of 16 of our toehold switches. The 16th toehold and specifically the part BBa\_K3727030 that corresponds to the Toehold\_Switch-1246 (3) was not cloned. We troubleshot to find out what could be the issue. Even though we received colonies on the plates there was no bacterial growth in our liquid cultures. For this reason, we tried preparing a new antibiotic stock, new plates with the proper antibiotic, and a new LB growth medium. However, the results were still disappointing. Since we didn’t have a lot of time to repeat the cloning procedures for this toehold we had the cloning of it at a stalemate until the end of the presentation.

## 3rd Direction

After we ensured that our toeholds functioned properly, we proceeded to the determination of the isothermal amplification method that would precede the detection of miRNAs by the Toehold Switches. To increase our method’s sensitivity and affordability, we decided to use the Exponential Amplification Reaction (EXPAR) method.

Our EXPAR protocols can be divided into 3 engineering cycles:

* **1.** Our attempts to establish an isothermal amplification protocol high specificity at 65 oC with end product DNA
* **2.** Our attempts to establish a low-temperature isothermal amplification protocol at 37 oC with end product DNA
* **3.** Our attempts to establish a low-temperature amplification protocol at 37 oC with end product RNA and the Template DNA needed for this reaction

### | Engineering cycle 1

**~Design**

The EXPAR technique requires instead of primers, a very specific single-stranded DNA sequence, the so-called “Template DNA”, composed of three main regions (Figure 10).

Two complementary with the microRNA sequences in both 3' and 5' end (black color) and a nicking site in the reverse orientation in the middle (green color). The reaction starts when the miRNA binds in the first region (3' end) and the DNA polymerase starts the replication. The nicking enzyme cuts the upper clone of the dsDNA in the nicking site and because the polymerase lacks the exonuclease activity a DNA sequence of the miRNA is released.

![](https://static.igem.org/mediawiki/2021/2/2a/T--Thessaloniki--engineering_1.png)

Figure 10: EXPAR Template DNA. The red color indicates the microRNA, the black areas that are complementary with the microRNA regions and the green color indicates the nicking site.

More specifically, we designed three Template DNA sequences, one for each of the selected microRNAs (Table 4).

| microRNA | Template DNA sequence | Size |
| --- | --- | --- |
| miR-30e-5p | 5'-CTTCCAGTCAAGGATGTTTACAGCAGTGCTTCCAGTCAAGGATGTTTACA-3P | 50 b |
| miR-143-3p | 5'-GAGCTACAGTGCTTCATCTCAGCAGTGGAGCTACAGTGCTTCATCTCA-3P | 48 b |
| miR-1246 | 5'-CCTGCTCCAAAAATCCATTGCAGTGCCTGCTCCAAAAATCCATT-3P | 44 b |

Table 4: The Template DNA sequences that we designed for each microRNA

We ordered the sequences from the Lab Supplies Company with phosphate-modified 3’ ends, for increased specificity [6].

As a nicking enzyme, we chose Nb. BtsI because its recognition sequence is not present in any of the other sequences used (plasmids, toeholds, eGFP gene). Moreover, according to NEB, the supplier of the enzyme, Nb. BtsI has good activity both at low and high temperatures, such as 37 oC and 65 oC, respectively [7].

Figure 11 shows the nicking site of Nb. BtsI. Inevitably, when the enzyme cuts the upper clone of the template DNA, the miRNA released has 5 extra bases “5’-CACTGC-3’” on its 3’ end that might affect its detection by the toeholds.

![](https://static.igem.org/mediawiki/2021/0/02/T--Thessaloniki--engineering_figure_11.png)

Figure 11: Nicking site of Nb.BtsI enzyme

Thus, we decided to check the thermodynamic characteristics of the reaction before we use them in the lab. The results based on Gibbs free energy showed that these extra bases on miRNAs increase the affinity with the toehold switches. Specifically, we calculated the binding energy of the two molecules, the miRNAs with the extra bases and the toehold switches, with the RNAcofold.exe of ViennaRNA software and we observed that the binding energy increased significantly in comparison with the miRNAs without the extra bases.

According to recent bibliography, while EXPAR is a cheap technique with very good amplification results, it has a lot of by-products mainly due to the ab initio DNA synthesis by the enzymes used [6].

Ab initio DNA synthesis is the synthesis of dsDNA from free dNTPs in the absence of template or miRNA. The dsDNA produced by this procedure consists of short (4–36 bp) tandem repeats of AT with a low GC-content [8]. But, when the reaction takes place at a high temperature, by-products seem to be reduced and the reaction specificity increases [8]. That’s why we chose the EXPAR protocol at 65oC to ensure higher specificity as it is higher than the melting temperature of our microRNAs (Table 5).

| miR | Sequence | Size | Tm\* |
| --- | --- | --- | --- |
| 30e-5p | UGUAAACAUCCUUGACUGGAAG | 22 | 45.9 °C |
| 143-3p | GGUGCAGUGCUGCAUCUCUGGU | 22 | 55.6 °C |
| 1246 | AAUGGAUUUUUGGAGCAGG | 19 | 42 °C |

Table 5: Melting temperature of the microRNAs used

\* The melting temperature was calculated with the type: Tm= (wA+xT) \* 2 + (yG+zC) \* 4

For our reaction, we chose Bst 2.0 DNA polymerase because according to the bibliography it has low ab initio synthesis and when used with Nb. BtsI works perfectly at 65oC and it has been used in EXPAR protocols with good results [9].

SYBR Green I was used to monitor the reaction in the qPCR machine (Applied Biosystems 7500 Fast Real-Time PCR System). SYBR Green I binding to dsDNA absorbs blue light (497 nm) and emits green light (520 nm) [10].

**~Build**

We followed the protocol of Ellie Mok et al. (2016) [11] and the reagents used are the following:

* ~Template DNA (instead of primers) for each microRNA
* ~microRNAs
* ~Nb.BtsI (nicking enzyme)
* ~rCutSmart™ Buffer (buffer for Nb.BtsI)
* ~Bst 2.0 DNA Polymerase
* ~Isothermal Amplification Buffer (buffer for Bst 2.0 DNA Polymerase)
* ~Deoxynucleotide (dNTP) Solution Mix
* ~SYBR Green I
* ~PCR H2O

Briefly, the procedure we completed is the following:

* **1.** Design of the protocol in the qPCR machine
* **2.** Template DNA and microRNAs preparation and aliquots
* **3.** Incubation of Template DNA and miRNA to achieve hybridization
* **4.** Adding of the enzymes
* **5.** Isothermal amplification and reaction monitoring in the qPCR machine
* **6.** Electrophoresis of the results

You can find the analytical protocol in our Experiments Page in the section "EXPAR":

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

**~Test**

When we tested the above-mentioned protocol we noticed that while our reaction was successful the amplification ratio was not as high as in the bibliography and that we had a lot of by-products even in the negative controls (samples with all the reagents except the miRNA).

We were very careful when we were creating the negative control and no miRNA was added into these samples, which means that ab initio DNA synthesis happened.

**~Learn**

Although initially, we wanted to test more conditions of the above-mentioned protocol, we decided not to proceed with an isothermal amplification method at 65 oC, because the high temperature used creates the need for expensive equipment. The rest of our reactions and mainly the toehold switches reactions are conducted at 37oC, which means that if the final product of our project needs 65oC for the amplification, it also needs a machine such as a PCR and that increases the cost.

So based on the first results of this isothermal amplification method, we chose to change our DNA polymerase with Klenow which works perfectly at 37 oC.

### | Engineering Cycle 2

**~Design**

We used the same template DNA and the only thing that changed in our design was the DNA polymerase; instead of Bst 2.0 DNA polymerase, we used Klenow Fragment (3'→5' exo-) which also lacks the exonuclease activity [12].

**~Build**

We followed again the same protocol of Ellie Mok et al. (2016) [11] and the reagents used are the following:

* ~Template DNA (instead of primers) for each microRNA
* ~microRNAs
* ~Nb.BtsI (nicking enzyme)
* ~rCutSmart™ Buffer (buffer for Nb.BtsI)
* ~Klenow Fragment (3'→5' exo-) (DNA polymerase)
* ~NEBuffer 2 (buffer for Klenow Fragment (3'→5' exo-))
* ~Deoxynucleotide (dNTP) Solution Mix
* ~SYBR Green I
* ~PCR H2O

The procedure we followed was the same, too. The only difference was the temperature of the reaction that in all cases, both in the hybridization and amplification step, was at 37 oC.

You can find the analytical protocol in our Experiments Page in the section "EXPAR":

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

**~Test**

Once again, when we tested our protocol we noticed that while our reaction was successful, the amplification ratio was ever lower compared to 65oC and that we had by-products even in the negative controls, too. We were very careful when we were creating the negative control samples, which means that ab initio DNA synthesis happened here, too.

We then noticed that the final concentration of Klenow Fragment DNA polymerase we used was lower than in the suggested protocol and that the total amount of Mg2+ in the final sample was high because both of the buffers used contained Mg2+ [13], [14].

Unfortunately, due to the small amount of Klenow Fragment DNA polymerase that we had in our possession and the large number of samples that we had to test, we couldn’t increase the final DNA polymerase concentration in the samples. Thus, we decided to lower the final concentration of NEBuffer 2 from 1x to 0.5x in an attempt to reduce the total concentration of Mg2+. We chose to reduce the buffer of the DNA polymerase and not the buffer of the nicking enzyme because according to NEB, Klenow Fragment (3'→5' exo-) has full functional activity when used with rCutSmart™ Buffer [15].

After these modifications, we noticed again ab initio DNA synthesis in the negative controls but the amplification ratio increased a little bit, and more copies of our miRNAs were produced.

**~Learn**

Although a small improvement of the protocol was achieved, more conditions should be tested. Unfortunately, due to the pressuring timeline of the competition, we didn’t have the time to order Klenow Fragment (3'→5' exo-) DNA polymerase to test our protocol with a bigger final concentration of the polymerase.

So, we decided to use the rest of the polymerase to test a slightly different EXPAR-like amplification protocol. The problem with EXPAR is that the final product produced is DNA while our toeholds are designed to detect RNA sequences. Thus we decided to modify a little bit the above-mentioned protocol to produce RNA sequences as a final product.

### | Engineering Cycle 3

**~Design**

This time we designed different "Template DNA" sequences, composed of three main regions (Figure 12); instead of a nicking site, they have a T7 RNA polymerase promoter sequence in the reverse orientation (blue region). The 5' complementary region is designed to be double-stranded (green region) to ensure that the miRNA can only bind to the 3' end of the template (black region), which makes the technique more sensitive.

![](https://static.igem.org/mediawiki/2021/b/be/T--Thessaloniki--engineering_2.png)

Figure 12: EXPAR-RNA Protocol Template DNA (the red color indicates the microRNA)

Only when the miRNA binds in the 3' end, a DNA polymerase can start the replication and a double-stranded DNA sequence of the T7 RNA polymerase promoter is produced. Then T7 RNA polymerase can start the transcription of the miRNA and a lot of RNA copies of the target miRNA are produced.

More specifically, we designed three Template DNA sequences, one for each of the selected microRNAs (with red color is the T7 promoter sequence):

| microRNA | Template DNA sequence | Size |
| --- | --- | --- |
| miR-30e-5p | 3P-GAAGGTCAGTTCCTACAAATGT-5' | 22 b |
|  | 5'-CTTCCAGTCAAGGATGTTTACACTATAGTGAGTCGTATTACTTCCAGTCAAGGATGTTTACA-3P | 62 b |
| miR-143-3p | 3P-CTCGATGTCACGAAGTAGAGT-5' | 21 b |
|  | 5'-GAGCTACAGTGCTTCATCTCACTATAGTGAGTCGTATTAGAGCTACAGTGCTTCATCTCA-3P | 60 b |
| miR-1246 | 3P-GGACGAGGTTTTTAGGTAA-5' | 19 b |
|  | 5'-CCTGCTCCAAAAATCCATTCTATAGTGAGTCGTATTACCTGCTCCAAAAATCCATT-3P | 56 b |

| Template DNA part | Modification of 3’ end | Sequence (5’ end to 3’ end) |
| --- | --- | --- |
| ER-143-3p-UP | phosphorylated | TGAGATGAAGCACTGTAGCTC |
| ER-143-3p-DOWN | NONE | GAGCTACAGTGCTTCAT |
| ER-143-3p-MAIN | phosphorylated | CTCACTATAGTGAGTCGTATTAGAGCTACAGTGCTTCATCTCA |
| ER-30e-5p-UP | phosphorylated | TGTAAACATCCTTGACTGGAAG |
| ER-30e-5p-DOWN | NONE | CTTCCAGTCAAGGATGTTT |
| ER-30e-5p-MAIN | phosphorylated | ACACTATAGTGAGTCGTATTACTTCCAGTCAAGGATGTTTACA |
| ER-1246-UP | Phosphorylated | AATGGATTTTTGGAGCAGG |
| ER-1246-DOWN | NONE | CCTGCTCCAAAAATCC |
| ER-1246-MAIN | phosphorylated | ATTCTATAGTGAGTCGTATTACCTGCTCCAAAAATCCATT |

**~Build:**

This time before we started with the amplification protocol, we had to “build” our Template DNA sequences in the lab. For this reason, we implemented a T4 ligase protocol based on NEB’s [4].

We then followed an EXPAR-like amplification protocol inspired by the work of Nicholas Emery et al.; [6]. The reagents we used are the following:

* ~Template DNA (instead of primers) for each microRNA (previously prepared with primer ligation T4 protocol)
* ~microRNAs
* ~T7 RNA Polymerase
* ~T7 RNA Polymerase Buffer
* ~Klenow Fragment (3'→5' exo-) (DNA polymerase)
* ~NEBuffer 2 (buffer for Klenow Fragment (3'→5' exo-))
* ~Deoxynucleotide (dNTP) Solution Mix
* ~Ribonucleotide (rNTP) Solution Mix
* ~SYBR Green I
* ~PCR H2O

Briefly, the procedure we completed is the following:

* **1.** Incubation of Template DNA “-up” and “-down” to achieve hybridization of the double-stranded region of the Template DNA sequence (at 51oC). We used the PROMEGA ™ Calculator https://worldwide.promega.com/resources/tools/biomath/tm-calculator/ to find the best temperature for the Template DNA “-up” and “-down” hybridization
* **2.** Ligation of the double-stranded part with the “-main” part of the Template DNA sequence with T4 DNA ligase (at 37oC)
* **3.** Deactivation of T4 ligase
* **4.** Design of the protocol in the qPCR machine
* **5.** Incubation of Template DNA and miRNA to achieve hybridization (at 37oC)
* **6.** Adding of the enzymes
* **7.** Isothermal amplification and reaction monitoring in the qPCR machine (at 37oC)
* **8.** Electrophoresis of the results

You can find both of the protocols in our Experiments Page in the section "EXPAR":

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

**~Test**

The first time we tried the ligation protocol was a total disaster. After a brainstorming session, we concluded that we should increase the incubation period for the T4 DNA ligase and that we should use a new T4 ligase buffer. T4 ligase buffer contains ATP that is sensitive to freeze-thaw cycles and without it, the T4 ligase has no activity.

Indeed, the use of a new buffer gave us better results and when we increased the incubation period at 37 oC we had an even better outcome.

Then we proceeded to the EXPAR - RNA amplification protocol. The first time was also a total disaster and no amplification happened.

Once again, we concluded that we should reduce the Mg2+ concentration because all of the buffers used (T4 ligase buffer, NEBuffer 2, and T7 RNA Polymerase Buffer) contained Mg2+, which in high amounts might inhibit the reaction [16].

Because the right Template DNA is necessary for the reaction, we decided not to change the amount of T4 ligase Buffer used.

Thus, we decided to lower the final concentration of NEBuffer 2 from 1x to 0.25x and the final concentration of T7 RNA polymerase buffer from 1x to 0.5x. We reduced the NEBuffer 2 more because the T7 RNA polymerase buffer contains spermidine which is very important for the reaction and is not present in the other two buffers.

After these modifications, our EXPAR-like RNA protocol succeeded, and as the continuously increased signal of SYBR Green I indicated. Nevertheless, in the reaction, there were again by-products as the diagram of the melting curve analysis indicates (Figure 13). If there weren’t any by-products we could only see one pick in the diagram.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Thessaloniki--curve_RNA.jpg)

Figure 13: Diagram of the melting curve analysis of the EXPAR - RNA Protocol products. With blue color, we have miR-30e-5p, with brown color miR-143-3p, and with grey color miR-1246.

**~Learn**

In the end, we managed to amplify our microRNAs at 37 oC with both EXPAR-DNA Protocol and EXPAR-RNA Protocol, too. So we were able to proceed with our Proof of Concept and to test the products of the two reactions with the best of our toehold switch sequences.

Nevertheless, more experiments need to be done to improve the amplification ratio so that we can achieve even bigger sensitivity.

We troubleshot to find out how to do that - e.g. different polymerase concentrations -, but unfortunately, we didn’t have the necessary time to test our hypothesis.

**We are determined to continue our experimental procedures after the wiki freeze but we hope that some of the future iGEM teams continue experimenting with our innovative EXPAR-RNA protocol, too!**

## Summary

* **1.** 2 Engineering cycles for improving the Toehold Switches’ detection
  + **i)**Replacement of the RBS
  + **ii)**Change of the reporter gene
* **2.** 2 Engineering cycles for effective cloning of our system with 2 different cloning procedures:
  + **i)**Use of the Biobrick Assembly
  + **ii)**Use of PCR cloning
* **3.** 3 Engineering cycles for effective isothermal amplification of our microRNAs with 3 different cloning procedures:
  + **i)**EXPAR-DNA isothermal amplification protocol at 65 oC with end product DNA
  + **ii)**EXPAR-DNA isothermal amplification protocol at 37 oC with end product DNA
  + **iii)**EXPAR-RNA isothermal amplification protocol at 37 oC with end product RNA

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* **|[12]** New England Biolabs, “Klenow Fragment (3'→5' exo-)”, Protocol, 2021, https://international.neb.com/products/m0212-klenow-fragment-3-5-exo#Product%20Information, (accessed Oct. 17, 2021)
* **|[13]** New England Biolabs, “NEBuffer™ 2”, 2021, https://international.neb.com/products/b7002-nebuffer-2#Product%20Information, (accessed Oct. 17, 2021)
* **|[14]** New England Biolabs, “rCutSmart™ Buffer”, 2021, https://international.neb.com/products/b6004-rcutsmart-buffer#Product%20Information, (accessed Oct. 17, 2021)
* **|[15]** New England Biolabs, “Activity of DNA Modifying Enzymes in rCutSmart™ Buffer”, Usage Guidelines, 2021, https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-dna-modifying-enzymes-in-cutsmart-buffer, (accessed Oct. 17, 2021)
* **|[16]** N. J. Emery, S. Majumder and A. P. Liu, “Synergistic and non-specific nucleic acid production by T7 RNA polymerase and Bsu DNA polymerase catalyzed by single-stranded polynucleotides”, Synthetic and Systems Biotechnology, vol. 3, no. 2, pp. 130-134, Jun. 2018

![](https://static.igem.org/mediawiki/2021/4/4a/T--Thessaloniki--logo_igem.png)

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[![](https://static.igem.org/mediawiki/2021/7/75/T--Thessaloniki--logo_aristotle.png)](https://www.auth.gr/en)

[![](https://static.igem.org/mediawiki/2021/e/e0/T--Thessaloniki--iGEM_logo_light.png)](https://2021.igem.org/Main_Page)

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[![](https://static.igem.org/mediawiki/2021/9/91/T--Thessaloniki--logo_idt.png)](https://eu.idtdna.com/pages)
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[![](https://static.igem.org/mediawiki/2021/d/d2/T--Thessaloniki--logo_biogenea.png)](https://biogenea.gr/)

* | Greece, Thessaloniki
* | Aristotle University
* | School of Sciences
* | Department of Biology

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)
* | igemthessaloniki@gmail.com

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

# Team:Thessaloniki/Experiments

☰
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×

* ## PROJECT
  + [| Description](https://2021.igem.org/Team%3AThessaloniki/Description)
  + [| Entrepreneurship](https://2021.igem.org/Team%3AThessaloniki/Entrepreneurship)
  + [| Model](https://2021.igem.org/Team%3AThessaloniki/Model)
  + [| Results](https://2021.igem.org/Team%3AThessaloniki/Results)
  + [| Software](https://2021.igem.org/Team%3AThessaloniki/Software)
  + [| Medals](https://2021.igem.org/Team%3AThessaloniki/Medals)
  + [| Hardware](https://2021.igem.org/Team%3AThessaloniki/Hardware)
  + [| Resources Table](https://2021.igem.org/Team%3AThessaloniki/Resources_Table)
  + [| Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)
  + [| Wiki Map](https://2021.igem.org/Team%3AThessaloniki/Wiki_Map)
  + [| Implementation](https://2021.igem.org/Team%3AThessaloniki/Implementation)
  + [| Judging Form](https://igem.org/2021_Judging_Form?id=3727)
  + [| Proof of Concept](https://2021.igem.org/Team%3AThessaloniki/Proof_Of_Concept)
  + [| Our Inspiration](https://2021.igem.org/Team%3AThessaloniki/Inspiration)
  + [| Contribution](https://2021.igem.org/Team%3AThessaloniki/Contribution)
* ## OUTREACH
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Human_Practices)
  + [| Collaborations](https://2021.igem.org/Team%3AThessaloniki/Collaborations)
  + [| Partnership](https://2021.igem.org/Team%3AThessaloniki/Partnership)
  + [| Communication](https://2021.igem.org/Team%3AThessaloniki/Communication)
  + [| Education](https://2021.igem.org/Team%3AThessaloniki/Education)
  + [| Promotion](https://2021.igem.org/Team%3AThessaloniki/Communication/Promotion)
* ## TEAM
  + [| PI](https://2021.igem.org/Team%3AThessaloniki/Team#PI)
  + [| Members](https://2021.igem.org/Team%3AThessaloniki/Team#Members)
  + [| Mentors](https://2021.igem.org/Team%3AThessaloniki/Team#mentors)
  + [| Advisors](https://2021.igem.org/Team%3AThessaloniki/Team#advisors)
  + [| Attributions](https://2021.igem.org/Team%3AThessaloniki/Attributions)
* ## NOTEBOOK
  + [| Team](https://2021.igem.org/Team%3AThessaloniki/Notebook)
  + [| Photo Album](https://2021.igem.org/Team%3AThessaloniki/Notebook/Photos)
  + [| Wet Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
  + [| Dry Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Dry)
  + [| FR](https://2021.igem.org/Team%3AThessaloniki/Notebook/FR)
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Notebook/Human)
* ## LAB
  + [| Parts](https://2021.igem.org/Team%3AThessaloniki/Parts)
  + [| Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
  + [| Safety](https://2021.igem.org/Team%3AThessaloniki/Safety)
* ## SPONSORS
  + [| Sponsors](https://2021.igem.org/Team%3AThessaloniki/Sponsors)
  + [| Become a Sponsor](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Become)
  + [| Special Thanks](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Thanks)

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)

igemthessaloniki@gmail.com

[* Solutions Preparation](#solutions)
[* Bacterial Cultures](#bacterial)
[* DNA Isolation](#iso)
[* DNA Amplicifation](#amp)
[* Electrophoresis](#elect)
[* Cloning](#cloning)
[* Cell-Free Protein Expression](#cell)
[* EXPAR](#expar)

## Experiments

**During our Wet Lab, we used a series of protocols for our experiments that helped us shape our project. In hope that these protocols might help future iGEM teams we attach them below!**

[![](https://static.igem.org/mediawiki/2021/f/fa/T--Thessaloniki--scroll_down.png)](#up)

## Preparing Solutions

[* #### Preparation of TE Buffer](https://static.igem.org/mediawiki/2021/3/36/T--Thessaloniki--Preparation_of_TE_Buffer.pdf)
[* #### Tris-HCI (1M) recipe](https://static.igem.org/mediawiki/2021/6/60/T--Thessaloniki--Tris-HCL_%281M%29_recipe.pdf)
[* #### Preparation of Glycerol 50%](https://static.igem.org/mediawiki/2021/e/e4/T--Thessaloniki--Preparation_of_Glycerol_50.pdf)

## Bacterial Cultures

[* #### Preparing LB-agar](https://static.igem.org/mediawiki/2021/7/76/T--Thessaloniki--Preparing_LB-agar.pdf)
[* #### Preparing LB-agar plates](https://static.igem.org/mediawiki/2021/5/56/T--Thessaloniki--Preparing_LB-agar_plates.pdf)
[* #### Preparation of Chemically Competent Cells](https://static.igem.org/mediawiki/2021/6/68/T--Thessaloniki--Preparation_of_Chemicallly_Competent_Cells.pdf)
[* #### Preparation of glycerol stock](https://static.igem.org/mediawiki/2021/7/79/T--Thessaloniki--Preparation_of_glycerol_stock.pdf)
[* #### Inoculation of liquid bacterial cultures](https://static.igem.org/mediawiki/2021/3/30/T--Thessaloniki--Inoculation_of_liquid_bacterial_cultures.pdf)
[* #### Streaking and Isolating Bacteria on an LB Agar Plate](https://static.igem.org/mediawiki/2021/d/d6/T--Thessaloniki--Streaking_and_Isolating_Bacteria_on_an_LB_Agar_Plate.pdf)

## DNA Isolation

[* #### Plasmid DNA Isolation with miniprep](https://static.igem.org/mediawiki/2021/e/eb/T--Thessaloniki--plasmid_dna_isolation_with_miniprep.pdf)
[* #### Gel Extraction](https://static.igem.org/mediawiki/2021/c/c4/T--Thessaloniki--gel_extraction.pdf)
[* #### PCR Cleanup](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--PCR_Cleanup.pdf)

## DNA Amplification

[* #### Primer Preparation](https://static.igem.org/mediawiki/2021/0/0f/T--Thessaloniki--primer_preparation.pdf)
[* #### PCR using Q5 High Fidelity](https://static.igem.org/mediawiki/2021/d/d2/T--Thessaloniki--PCR_using_Q5_High_Fidelity.pdf)

## Electrophoresis

[* #### Preparation of 10x TAE buffer](https://static.igem.org/mediawiki/2021/6/69/T--Thessaloniki--preparation_of_10x_TAE_buffer.pdf)
[* #### Electrophoresis](https://static.igem.org/mediawiki/2021/8/86/T--Thessaloniki--electrophoresis.pdf)

## Cloning

[* #### Digestion of g-Blocks](https://static.igem.org/mediawiki/2021/e/e5/T--Thessaloniki--digestion_of_g-blocks.pdf)
[* #### Digestion of pSB1C3 Vector](https://static.igem.org/mediawiki/2021/d/dc/T--Thessaloniki--digestion_of_pSB1C3_vector.pdf)
[* #### Dephosphorylation of linearized vector](https://static.igem.org/mediawiki/2021/1/1b/T--Thessaloniki--dephosphorylation_of_linearized_vector.pdf)
[* #### Ligation with T4 ligase](https://static.igem.org/mediawiki/2021/f/fa/T--Thessaloniki--Ligation_with_T4_ligase.pdf)
[* #### Transformation of competent cells](https://static.igem.org/mediawiki/2021/b/b7/T--Thessaloniki--transformation_of_competent_cells.pdf)
[* #### Diagnostic Restriction Digestions](https://static.igem.org/mediawiki/2021/0/04/T--Thessaloniki--diagnostic_restriction_digestions.pdf)
* #### ------------
[* #### PCR cloning](https://static.igem.org/mediawiki/2021/b/b8/T--Thessaloniki--PCR_cloning.pdf)
* #### ------------

## Cell-Free Protein Expression

[* #### Resuspension of gBlocks](https://static.igem.org/mediawiki/2021/9/91/T--Thessaloniki--resuspension_of_gBlocks.pdf)
[* #### In vitro Expression with Cell Free System](https://static.igem.org/mediawiki/2021/d/d2/T--Thessaloniki--In_vitro_Expression_with_Cell_Free_System.pdf)

## EXPAR

[* #### Primer Ligation with T4](https://static.igem.org/mediawiki/2021/3/3e/T--Thessaloniki--Primer_Ligation_with_T4.pdf)
[* #### EXPAR DNA Protocol 37 ℃](https://static.igem.org/mediawiki/2021/0/02/T--Thessaloniki--EXPAR_DNA_Protocol_37oC.pdf)
[* #### EXPAR DNA Protocol 65 ℃](https://static.igem.org/mediawiki/2021/7/75/T--Thessaloniki--EXPAR_DNA_Protocol_65oC.pdf)
* #### ------------
[* #### EXPAR RNA Protocol 37 ℃](https://static.igem.org/mediawiki/2021/6/66/T--Thessaloniki--EXPAR_RNA_Protocol_37oC.pdf)
* #### ------------

![](https://static.igem.org/mediawiki/2021/4/4a/T--Thessaloniki--logo_igem.png)

[![](https://static.igem.org/mediawiki/2021/2/26/T--Thessaloniki--logo_rgcc.png)](https://www.rgcc-group.com/)

[![](https://static.igem.org/mediawiki/2021/9/9a/T--Thessaloniki--logo_pfizer.png)](https://www.pfizer.gr/)
[![](https://static.igem.org/mediawiki/2021/3/35/T--Thessaloniki--logo_unifarma.png)](https://www.uni-pharma.gr/)

[![](https://static.igem.org/mediawiki/2021/7/75/T--Thessaloniki--logo_aristotle.png)](https://www.auth.gr/en)

[![](https://static.igem.org/mediawiki/2021/e/e0/T--Thessaloniki--iGEM_logo_light.png)](https://2021.igem.org/Main_Page)

[![](https://static.igem.org/mediawiki/2021/c/ca/T--Thessaloniki--logo_antisel.png)](https://antisel.gr/)
[![](https://static.igem.org/mediawiki/2021/c/c3/T--Thessaloniki--logo_benchling.png)](https://www.benchling.com/)
[![](https://static.igem.org/mediawiki/2021/4/49/T--Thessaloniki--logo_bioline.jpg)](https://k2web3.eu/)

[![](https://static.igem.org/mediawiki/2021/9/9b/T--Thessaloniki--logo_neb.png)](https://international.neb.com/)
[![](https://static.igem.org/mediawiki/2021/a/a1/T--Thessaloniki--logo_farma.png)](https://webtv.fsth.gr/)
[![](https://static.igem.org/mediawiki/2021/f/f8/T--Thessaloniki--logo_ELPEN.png)](https://www.elpen.gr/)

[![](https://static.igem.org/mediawiki/2021/9/91/T--Thessaloniki--logo_idt.png)](https://eu.idtdna.com/pages)
[![](https://static.igem.org/mediawiki/2021/b/b6/T--Thessaloniki--logo_rct.png)](https://rctech.com/)
[![](https://static.igem.org/mediawiki/2021/d/d2/T--Thessaloniki--logo_biogenea.png)](https://biogenea.gr/)

* | Greece, Thessaloniki
* | Aristotle University
* | School of Sciences
* | Department of Biology

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)
* | igemthessaloniki@gmail.com

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

# Team:Thessaloniki/Results

☰
[![](https://static.igem.org/mediawiki/2021/2/21/T--Thessaloniki--menu_icon.png)](https://2021.igem.org/Team%3AThessaloniki)

×

* ## PROJECT
  + [| Description](https://2021.igem.org/Team%3AThessaloniki/Description)
  + [| Entrepreneurship](https://2021.igem.org/Team%3AThessaloniki/Entrepreneurship)
  + [| Model](https://2021.igem.org/Team%3AThessaloniki/Model)
  + [| Results](https://2021.igem.org/Team%3AThessaloniki/Results)
  + [| Software](https://2021.igem.org/Team%3AThessaloniki/Software)
  + [| Medals](https://2021.igem.org/Team%3AThessaloniki/Medals)
  + [| Hardware](https://2021.igem.org/Team%3AThessaloniki/Hardware)
  + [| Resources Table](https://2021.igem.org/Team%3AThessaloniki/Resources_Table)
  + [| Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)
  + [| Wiki Map](https://2021.igem.org/Team%3AThessaloniki/Wiki_Map)
  + [| Implementation](https://2021.igem.org/Team%3AThessaloniki/Implementation)
  + [| Judging Form](https://igem.org/2021_Judging_Form?id=3727)
  + [| Proof of Concept](https://2021.igem.org/Team%3AThessaloniki/Proof_Of_Concept)
  + [| Our Inspiration](https://2021.igem.org/Team%3AThessaloniki/Inspiration)
  + [| Contribution](https://2021.igem.org/Team%3AThessaloniki/Contribution)
* ## OUTREACH
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Human_Practices)
  + [| Collaborations](https://2021.igem.org/Team%3AThessaloniki/Collaborations)
  + [| Partnership](https://2021.igem.org/Team%3AThessaloniki/Partnership)
  + [| Communication](https://2021.igem.org/Team%3AThessaloniki/Communication)
  + [| Education](https://2021.igem.org/Team%3AThessaloniki/Education)
  + [| Promotion](https://2021.igem.org/Team%3AThessaloniki/Communication/Promotion)
* ## TEAM
  + [| PI](https://2021.igem.org/Team%3AThessaloniki/Team#PI)
  + [| Members](https://2021.igem.org/Team%3AThessaloniki/Team#Members)
  + [| Mentors](https://2021.igem.org/Team%3AThessaloniki/Team#mentors)
  + [| Advisors](https://2021.igem.org/Team%3AThessaloniki/Team#advisors)
  + [| Attributions](https://2021.igem.org/Team%3AThessaloniki/Attributions)
* ## NOTEBOOK
  + [| Team](https://2021.igem.org/Team%3AThessaloniki/Notebook)
  + [| Photo Album](https://2021.igem.org/Team%3AThessaloniki/Notebook/Photos)
  + [| Wet Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
  + [| Dry Lab](https://2021.igem.org/Team%3AThessaloniki/Notebook/Dry)
  + [| FR](https://2021.igem.org/Team%3AThessaloniki/Notebook/FR)
  + [| Human Practices](https://2021.igem.org/Team%3AThessaloniki/Notebook/Human)
* ## LAB
  + [| Parts](https://2021.igem.org/Team%3AThessaloniki/Parts)
  + [| Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
  + [| Safety](https://2021.igem.org/Team%3AThessaloniki/Safety)
* ## SPONSORS
  + [| Sponsors](https://2021.igem.org/Team%3AThessaloniki/Sponsors)
  + [| Become a Sponsor](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Become)
  + [| Special Thanks](https://2021.igem.org/Team%3AThessaloniki/Sponsors/Thanks)

* [![](https://static.igem.org/mediawiki/2021/c/cc/T--Thessaloniki--logo_in.png)iGEM Thessaloniki](https://www.linkedin.com/company/igem-thessaloniki)
* [![](https://static.igem.org/mediawiki/2021/2/24/T--Thessaloniki--logo_fb.svg)@iGEMthessaloniki](https://www.facebook.com/iGEMthessaloniki)
* [![](https://static.igem.org/mediawiki/2021/5/55/T--Thessaloniki--logo_insta.png)igem.thessaloniki](https://www.instagram.com/igem.thessaloniki/)

igemthessaloniki@gmail.com

[* Overview](#Overview)
[* Cloning Segment](#cloning)
[* Toehold Sector](#toe)
[* EXPAR Procedure](#expar)
[* References](#ref)

## Results

[![](https://static.igem.org/mediawiki/2021/f/fa/T--Thessaloniki--scroll_down.png)](#up)

## Overview

This year we wanted to make a difference in the lives of people with pancreatic cancer by implementing "METIS"; a MicroRNA Expression Toehold Investigation System. To make METIS a reality, we first had to prove that our method works. To do so, we conducted experiments to validate both our toehold switches and our Isothermal Amplification method. In this context, we divided our wet-lab experiments into 3 categories: the **Cloning Segment**, the **Toehold Sector**, and the **EXPAR Procedure**. In the following text, you can see our results that we can surely say are not only encouraging but rewarding, too.

For more information about the experiments we conducted, you can check our "Experiments" page and our "Wet Lab Notebook" page. You can also check out our "Engineering" page to see how we designed our experiments as a whole.

[* ##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
[* ##### Wet Lab Notebook](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)
[* ##### Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)

## Cloning Segment

After designing and ordering our parts, we immediately started the cloning procedure. You can find more information about our protocols and experiments on the following pages:

[* ##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)
[* ##### Wet Lab Notebook](https://2021.igem.org/Team%3AThessaloniki/Notebook/Wet)

Initially, we tried to clone our 16 sequences with the Biobrick Assembly method. Unfortunately, our efforts were for naught since we were not able to clone our parts using this technique. We constantly received empty plates, so we decided to troubleshoot. In the end, we chose a different technique and we succeeded in cloning 15 of 16 of our toeholds using PCR Cloning.

In the following photographs, you can see the results of the diagnostic restrictions electrophoresis:

* ![](https://static.igem.org/mediawiki/2021/a/a7/T--Thessaloniki--electrophoresis_1.jpg)

  Figure 1
* ![](https://static.igem.org/mediawiki/2021/b/ba/T--Thessaloniki--electrophoresis_2.png)

  Figure 2
* ![](https://static.igem.org/mediawiki/2021/9/96/T--Thessaloniki--electrophoresis_3.png)

  Figure 3
* ![](https://static.igem.org/mediawiki/2021/8/8c/T--Thessaloniki--electrophoresis_4.jpg)

  Figure 4
* ![](https://static.igem.org/mediawiki/2021/f/f4/T--Thessaloniki--electrophoresis_5.png)

  Figure 5
* ![](https://static.igem.org/mediawiki/2021/0/0c/T--Thessaloniki--electrophoresis_6.png)

  Figure 6
* ![](https://static.igem.org/mediawiki/2021/b/bd/T--Thessaloniki--electrophoresis_7.jpg)

  Figure 7

X’ = means uncut plasmid
Ε = means empty well

Specifically, the coding system for our toeholds is the following (Table 1):

|  |  |  |  |
| --- | --- | --- | --- |
| A1 = 143-3p-1 | B1 = 30e-5p-1 | B5 = 30e-5p-5 | C1 = 1246-1 |
| A2 = 143-3p-2 | B2 = 30e-5p-2 | B6 = 30e-5p-6 | C2 = 1246-2 |
| A3 = 143-3p-3 | B3 = 30e-5p-3 | B7 = 30e-5p-7 | - |
| A4 = 143-3p-4 | B4 = 30e-5p-4 | B8 = 30e-5p-8 | C4 = 1246-4 |

Table 1: Coding name for the toehold switches

In some cases, we digested the plasmids only with one enzyme, because the amount of the second enzyme was insufficient. In the pictures that correspond to these experiments, we can see that the size of some of the linearized plasmids is between 3-4 kb, which is the desired size. This means that the plasmid contains the insert and, hence, the cloning was successful (Figure 1, Figure 2, Figure 3, and Figure 4).

In the rest of the experiments, we used both of the two enzymes and in the corresponding pictures, you can see a band approximately at 1 kb. This is the size of the desired insert and we can conclude that the cloning procedure was successful (Figure 5, Figure 6, and Figure 7).

Our cloning results are summarized in Table 2, where you can see the name of the sample and its concentration.

| Code | Sample | Concentration |
| --- | --- | --- |
| A1 | T-143-3p-1 | 16.4 ng/ul |
| A2 | T-143-3p-2 | 158.0 ng/ul |
| A3 | T-143-3p-3 | 101.9 ng/ul |
| A4 | T-143-3p-4 | 144.7 ng/ul |
| B1 | T-30e-5p-1 | 57.1 ng/ul |
| B2 | T-30e-5p-2 | 56.3 ng/ul |
| B3 | T-30e-5p-3 | 34.0 ng/ul |
| B4 | T-30e-5p-4 | 38.3 ng/ul |
| B5 | T-30e-5p-5 | 108.1 ng/ul |
| B6 | T-30e-5p-6 | 132.5 ng/ul |
| B7 | T-30e-5p-7 | 66.6 ng/ul |
| B8 | T-30e-5p-8 | 98.6 ng/ul |
| C1 | T-1246-1 | 140,2 ng/ul |
| C2 | T-1246-2 | 57.9 ng/ul |
| C3 | T-1246-3 | - |
| C4 | T-1246-4 | 122.5 ng/ul |

Table 2: Cloning results of the samples

## Toehold Sector

Since we received our toehold switches, we began to work towards the *in vitro* protein synthesis assay. These experiments are the main part of our project and are critical for the implementation of our detection technology to ensure that our system functions properly. For our experiments, we used a cell-free system, and especially, we used the commercial PURExpress kit from New England Biolabs (NEB), (ΝΕΒ #Ε8600) which is highly purified and reliable for our proof of concept experiments. The highlights of these experiments are analyzed below.

**~Testing our toeholds with the cell-free system**

After the dry lab had designed from scratch 16 toehold switches (4 for hsa-miR-143-3p, 4 for hsa-miR-1246, and 8 for hsa-miR-30e-5p), the wet lab had to test their functionality. The key to functionality was the miRNA mimics (Thermofisher - mirVana® miRNA mimic #4464066) and eGFP (enhanced Green Fluorescent Protein). To test our toeholds, we used miRNA mimics and not urine from patients, because biological samples can be dangerous and difficult to handle. Moreover, in the urine, a lot of different microRNAs are present that could affect our results. So, before going into further detail, we first had to ensure that our toeholds work properly. For our experiments, we used the fluorescence plate reader “GloMax-Multi Detection System” for measuring the excitation at 488 nm and emission at 507 nm.

First, we wanted to ensure that our system functions properly and check whether there is a leakage signal. To do so, we used the PURExpress Protein Synthesis kit (ΝΕΒ #Ε6800). On the wells of the plate, we loaded:

* **1.** The toehold switches with the reagents of NEB - PURExpress® "*In Vitro* Protein Synthesis Kit" (#E8600).
* **2.** The toehold switches, as described above, with the complementary miRNA mimic (Thermofisher - mirVana® miRNA mimic #4464066).
* **3.** The reagents of the cell-free system NEB - PURExpress® “*In Vitro* Protein Synthesis Kit” (#E8600), as a negative control.
* **4.** The reagents of the cell-free system NEB - PURExpress® and the DHFR plasmid as a positive control, in case of electrophoresis.

The reagents used in the experiments are in Table 3:

| Reagents | Volume |
| --- | --- |
| Solution A | 10 ul |
| Solution B | 7.5 ul |
| RNAse inhibitor | 0.5 ul |
| Template DNA | 30 ng |
| miRNA | 10 ul from 10 nM |
| Nuclease free water | to 50 ul |

Table 3: Reagents used in the fluorescence experiments of toehold switches

The reaction was carried out up to 25 ul or 50 ul and the plate was incubated for up to 2 hours at 37℃.

The results we received were very promising for the toehold switch T-143-3p-1 (BBa\_K3727016) of hsa-miR-143-3p, the toehold switch T-30e-5p-8 (BBa\_K3727027) of hsa-miR-30e-5p, and finally the toehold switch T-1246-1 (BBa\_K3727028) of hsa-miR-1246.

![](https://static.igem.org/mediawiki/2021/3/3e/T--Thessaloniki--results_1.png)

Figure 8: Τhis graph presents the fluorescence results from toehold switches in the presence of miR-143-3p mimic (Thermofisher - mirVana® miRNA mimic #4464066) and without miR-143-3p, as a negative control. For each toehold switch, the first bar corresponds to the fluorescence results of the toehold switch in the presence of miR-143-3p and the second one shows the fluorescence results in the absence of miR-143-3p (Negative Control).

As seen in Figure 8, the toehold switch T-143-3p-1 (BBa\_K3727016) and the toehold switch T-143-3p-3 (BBa\_K3727018) function properly and are stable enough without leakage signal.

![](https://static.igem.org/mediawiki/2021/9/91/T--Thessaloniki--results_2.png)

Figure 9: Τhis graph presents the fluorescence results from the toehold switches in the presence of miR-30e-5p mimic (Thermofisher - mirVana® miRNA mimic #4464066) and without miR-30e-5p, as a negative control. For each toehold switch, the first bar corresponds to the fluorescence results of the toehold switch in the presence of miR-30e-5p and the second one shows the fluorescence results in the absence of miR-30e-5p (Negative Control).

As seen in Figure 9, the toehold switch T-30e-5p-8 (BBa\_K3727027) and the toehold switch T-30e-5p-6 (BBa\_K3727025) function properly and are stable enough without leakage signal.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Thessaloniki--results_3.png)

Figure 10: Τhis graph presents the fluorescence results from toehold switches in the presence of miR-1246 mimic (Thermofisher - mirVana® miRNA mimic #4464066) and without miR-1246, as a negative control. For each toehold switch, the first bar corresponds to the fluorescence results of the toehold switch in the presence of miR-1246 and the second one shows the fluorescence results in absence of miR-1246 (Negative Control).

As seen in Figure 10, the toehold switch T-1246-1 (BBa\_K3727028) and the toehold switch T-1246-4 (BBa\_K3727031) function properly and are stable enough without leakage signal.

It is worth mentioning that due to a problem with the miRNA-1246 and the company from which we bought it, we had to postpone those experiments. Unfortunately, when we were able to use the right miRNA, our fluorescent plate reader had a problem and we had to replace it with another one. Thus, the measurements are not similar.

**~Experiments of Quantification**

For our proof of concept, we checked the sensitivity of our system by quantifying the fluorescence produced by eGFP when different quantities of each miR are used. To achieve this, we executed experiments in black 96-well-plates (Greiner #655077) and used the plate reader “GloMax-Multi Detection System”, measuring the excitation at 488 nm and emission at 507 nm. We tested the same quantity of toehold switches with different quantities of each miR from 1 nM to 1 uM in different wells. As a negative control, we used in a well only the reagents of PURExpress “*In vitro* Protein Synthesis Kit” (NEB #6800) and in another well the reagents with the toehold switch in the absence of miR. For all the experimental procedures, we used miRNA mimics (Thermofisher - mirVana® miRNA mimic #4464066) which are complementary to each toehold switch. The reagents used in the experiments are in Table 4.

| Reagents | Volume |
| --- | --- |
| Solution A | 10 ul |
| Solution B | 7.5 ul |
| RNAse inhibitor | 0.5 ul |
| Template DNA | 30 ng |
| Nuclease free water | to 50 ul |

Table 4: Reagents used in the fluorescence experiments of toehold switches

The reaction was carried out up to 25 ul or 50 ul and the plate was incubated for up to 4 hours at 37℃. The results we received were very promising for the toehold switch T-143-3p-1 (BBa\_K3727016) of hsa-miR-143-3p, the toehold switch T-30e-5p-8 (BBa\_K3727027) of hsa-miR-30e-5p, and finally the toehold switch T-1246-1 (BBa\_K3727028) of hsa-miR-1246.

By observing Figure 11, Figure 12, and Figure 13, we concluded that the detected fluorescence of every toehold switch depends on the initial amount of the corresponding miRNA. Specifically, it is concluded that by increasing the amount of the miRNA, the fluorescence is increased too. The mathematical relationship between the fluorescence and the concentration of the miRNA is logarithmic for every system.

![](https://static.igem.org/mediawiki/2021/a/a4/T--Thessaloniki--results_4.png)
![](https://static.igem.org/mediawiki/2021/5/51/T--Thessaloniki--results_5.png)

Figure 11: This graph shows the fluorescence produced from the toehold switch T-143-3p-1 (BBa\_K3727016) of hsa-miR-143-3p according to the quantities of miR-143-3p from 10nM to 1000nM.

![](https://static.igem.org/mediawiki/2021/f/f3/T--Thessaloniki--results_6.png)
![](https://static.igem.org/mediawiki/2021/6/6f/T--Thessaloniki--results_7.png)

Figure 12: This graph shows the fluorescence produced from the toehold switch T-30e-5p-8 (BBa\_K3727027) of hsa-miR-30e-5p according to the quantities of miR-30e-5p-8 from 10nM to 1000nM.

![](https://static.igem.org/mediawiki/2021/0/0a/T--Thessaloniki--results_8.png)
![](https://static.igem.org/mediawiki/2021/3/31/T--Thessaloniki--results_9.png)

Figure 13: This graph shows the fluorescence produced from the toehold switch T-1246-1 (BBa\_K3727028) of hsa-miR-1246 according to the quantities of miR-1246 from 10nM to 1000nM.

## EXPAR Procedure

In the urine of both healthy individuals and patients with PDAC, miRNAs are in very low concentration. Thus, to increase the sensitivity of our method, we had to use an amplification technique to amplify the miRNAs we want to detect. The technique we chose to use is EXPAR an isothermal amplification method and our results concerning this part were very encouraging and promising, although, due to the lack of time, more needs to be done.

Firstly, we used an EXPAR protocol that amplifies our microRNAs at 65 ℃(you can find more information on our Experiments Page).

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

The results were very good and as both the Amplification Plot and the Gel Electrophoresis indicated that all of our microRNAs were amplified and their concentration increased up to x10^2 (negative control signal is max ~10,000 –Figure 15– while at the end of the method the signal of the rest samples is ~1,000,000 –Figure 14–).

![](https://static.igem.org/mediawiki/2021/0/0b/T--Thessaloniki--expar_1.jpg)

Figure 14: Amplification plot without negative controls for EXPAR – DNA Protocol at 65 ℃.

![](https://static.igem.org/mediawiki/2021/8/8e/T--Thessaloniki--results_10.jpg)

Figure 15: Amplification plot with negative controls for EXPAR – DNA Protocol at 65 ℃.

\*The negative controls contain only the DNA template and no microRNA.

Although our EXPAR protocol at 65 ℃ can be considered successful, we chose not to be our final amplification method. The reactions of our cell-free system should be conducted at 37 ℃ and if the amplification happens at 65 ℃ there will be a need for expensive equipment, that could reduce and increase the temperature at an instant, such as a PCR machine. We wanted our method to be inexpensive and thus we chose to proceed with the EXPAR protocol at 37 ℃.

The results of the EXPAR protocol at 37 ℃ weren’t that good as the results at 65 ℃, but they were still promising as the concentration of our microRNAs increased up to x10 based on the SYBR Green I signal (negative control signal is max ~1,000 –Figure 17– while at the end of the method the signal of the rest samples is ~10,000 –Figure 16–).

![](https://static.igem.org/mediawiki/2021/f/f9/T--Thessaloniki--expar_2.jpg)
![](https://static.igem.org/mediawiki/2021/0/09/T--Thessaloniki--expar_explain.png)

Figure 16: Amplification plot without negative controls for EXPAR – DNA Protocol at 37 ℃.

![](https://static.igem.org/mediawiki/2021/1/1a/T--Thessaloniki--expar_3.jpg)
![](https://static.igem.org/mediawiki/2021/0/09/T--Thessaloniki--expar_explain.png)

Figure 17: Amplification plot with negative controls\* for EXPAR – DNA Protocol at 37 ℃.

\*The negative controls contain only the DNA template and no microRNA.

Although, the results of the EXPAR – DNA Protocol at 37 ℃ were promising the final product of the amplification was DNA, but our toeholds are designed to detect RNA and not DNA. So we decided to change our protocol a little bit and to design new Template DNA sequences so that the final product of the amplification method to be RNA instead of DNA. In Figure 18, there is the final form of the Template DNA for the so-called EXPAR – RNA Protocol at 37 ℃.

![](https://static.igem.org/mediawiki/2021/1/13/T--Thessaloniki--res_expar.png)

Figure 18: The Template DNA designed for the EXPAR – RNA Protocol at 37 ℃

\*With green color is the up-template part, with purple color is the down-template part and with black color is the main-template part.

We ordered the three parts separately and to achieve the final form, we had to use the Primer Ligation with T4 Protocol. You can find more information on our Experiments Page:

[##### Experiments](https://2021.igem.org/Team%3AThessaloniki/Experiments)

More specifically, we wanted to ligate the dsDNA (composed from the up-and-down- template) with the ssDNA (main template). The first experiments with T4 ligase were a total disaster. But when we choose to use a new T4 Ligase Buffer for our experiments and when we increased the incubation time, we got satisfactory results (Figure 19). T4 ligase buffer contains ATP which is necessary for the T4 ligase and is very sensitive to temperature and repeated freeze-thaw cycles affect the stability of ATP according to NEB [1]. Probably, in our first buffer, the ATP was damaged and the T4 ligase did not work properly.

![](https://static.igem.org/mediawiki/2021/9/90/T--Thessaloniki--expar_ligation_1.jpg)

Figure 19: Electrophoresis results of Primer ligation with T4 protocol in 4% agarose gel (30 min)

After the successful ligation, we proceeded with the EXPAR – RNA Protocol at 37 ℃. Once again our first attempt was a total disaster (Figure 20) and no amplification happened, as the very high signal of the negative controls indicates.

![](https://static.igem.org/mediawiki/2021/2/29/T--Thessaloniki--expar_4.jpg)

Figure 20: Amplification plot with negative controls for EXPAR – RNA Protocol at 37 ℃.

We thought that the above-mentioned disaster was due to the high concentration of Mg2+ that was used. As shown in Table 5, all the buffers used contain Mg2+. Mg2+ is necessary for the enzymes to work, but a very high concentration can lead to the inhibition of the reaction, too [2].

|  | 1x NEBuffer 2 | 1x T7 RNA Polymerase Buffer | 1x rCutSmart™ Buffer | 1X T4 DNA Ligase Reaction Buffer |
| --- | --- | --- | --- | --- |
| Common components | For Klenow Fragment (3'→5' exo-) | For T7 RNA Polymerase | For Nb.BtsI - nicking enzyme | For T4 DNA Ligase |
| 10 mM MgCl2 | 6 mM MgCl2 | 10 mM Magnesium Acetate | 10 mM MgCl2 |
| 10 mM Tris-HCl | 40 mM Tris-HCl | 20 mM Tris-acetate | 50 mM Tris-HCl |
| 1 mM DTT | 1 mM DTT | - | 10 mM DTT |
| Other Components | 50 mM NaCl | 2 mM spermidine | 50 mM Potassium Acetate | 1 mM ATP |
| - | - | 100 µg/ml Recombinant Albumin | - |
| pH | pH 7.9@25°C | (pH 7.9 @ 25°C) | pH 7.9@25°C | (pH 7.5 @ 25°C) |

Table 5: Components of all the Buffers used in EXPAR experiments at 37 ℃, according to https://international.neb.com/

First, we thought that we should reduce the number of buffers used, to reduce the total Mg2+ concentration of the reaction. While in all buffers there are common components, there are different components, too, so we decided not to remove any of them from the reaction but to reduce a little the quantity of most of them in both EXPAR – RNA and DNA Protocol at 37 ℃.

After the above-mentioned changes, both of the Protocols worked better (Figure 21). The EXPAR – DNA Protocol showed less signal in the negative controls (Figure 21. A) while in EXPAR – RNA Protocol the concentration of the microRNAs increased up to x10-10^2 based on the SYBR Green I signal (negative control signal is max ~1,000, while at the end of the method the signal of the rest of the samples is >10,000 –Figure 21. B–).

![](https://static.igem.org/mediawiki/2021/5/50/T--Thessaloniki--expar_5.jpg)
![](https://static.igem.org/mediawiki/2021/f/f9/T--Thessaloniki--expar_6.jpg)
![](https://static.igem.org/mediawiki/2021/d/d5/T--Thessaloniki--expar_6a.jpg)

Figure 21: Amplification plots with negative controls for EXPAR - DNA (A) and EXPAR - RNA (B) Protocol at 37 ℃. Top (A): Amplification plot with negative controls for EXPAR - DNA Protocol at 37 ℃, Bottom (B): Amplification plot with negative controls for EXPAR - RNA Protocol at 37 ℃

In both reactions, miR-1246 had the best results, with a high signal in the presence of miR (~100,000) and a low signal in the negative control (between 1,000 and 10,000) (Figure 22).

![](https://static.igem.org/mediawiki/2021/c/c3/T--Thessaloniki--expar_7.jpg)

Figure 22: Amplification plot with negative control for miR-1246 after EXPAR - RNA Protocol at 37 ℃

\*With black color is the signal from the sample with miR-1246, while with red color is the signal of the negative control without the miR-1246.

After the promising results of the amplification protocols, we had to test the amplification products with the best of our toehold switches. The results were very good and confirmed the results from the amplification plots (Table 6). Once again, miR-1246 had the best results; signal from EXPAR-DNA miR sample: 130 vs signal from EXPAR-DNA negative control: 100. Specifically, in EXPAR-DNA products the miR-1246 concentration increased from 10 nM to probably more than 400 nM, indicating more than x10 amplification. As expected, in EXPAR-RNA, miR-30e and miR-1246 showed the best results; 150 with miR-30e vs 110 without and 190 with miR-1246 vs 160 without, respectively. But, unfortunately, we can’t confirm the exact increase.

The first two samples of each column of Table 6 were used as positive controls. The first line is the positive control for the EXPAR-RNA protocol as it contains a specific quantity of each miRNA. The second line is the positive control for the EXPAR-DNA protocol as it contains a specific quantity of each miRNA in the form of DNA with the extra 5 nucleotides in the 3’ end. For more information check our Engineering Page in the “3rd Direction” in the design of the first engineering cycle.

[##### Engineering](https://2021.igem.org/Team%3AThessaloniki/Engineering)

In contrast with our theoretical model, the insertion of the 5 extra nucleotides did not exhibit better fluorescence results.

| miR-30e Samples | Fluorescence | miR-143 Samples | Fluorescence | miR1246 - Samples | Fluorescence |
| --- | --- | --- | --- | --- | --- |
| miR-30e (2 nM) | 95 | miR-143 (2 nM) | 110 | miR-1246 (2 nM) | 98 |
| DNA of miR-30e (400 nM) | 100 | DNA of miR-143 (400 nM) | 99 | DNA of miR-1246 (400 nM) | 92 |
| ed miR-30e (x nM) | 120 | ed miR-143 (x nM) | 120 | ed miR-1246 (x nM) | 130 |
| ed miR-30e negative control (0 nM) | 110 | ed miR-143 negative control (0 nM) | 110 | ed miR-1246 negative control (0 nM) | 100 |
| er miR-30e (x nM) | 150 | er miR-143 (x nM) | 140 | er miR-1246 (x nM) | 190 |
| er miR-30e negative control (0 nM) | 110 | er miR-143 negative control (0 nM) | 150 | er miR-1246 negative control (0 nM) | 160 |
| Empty well | 95 | Empty well | 94 | Empty well | 93 |
| Empty well | 95 | Empty well | 94 | Empty well | 95 |
| Empty well | 95 | Empty well | 94 | Empty well | 93 |
| Empty well | 96 | Empty well | 94 | Empty well | 93 |
| Empty well | 95 | Empty well | 94 | Empty well | 94 |
| Empty well | 95 | Empty well | 94 | Empty well | 94 |

Table 6: Fluorescence results from the experiments with toehold switches and EXPAR products at 37 ℃.

\*Abbreviations: ed = EXPAR - DNA amplification protocol products | er = EXPAR - RNA amplification protocol products | x nM = unknown concentration

In all cases, the negative controls from EXPAR had higher fluorescence signals than the positive controls. This is due to the presence of SYBR Green I in the EXPAR samples. SYBR Green I was used for quantifying the EXPAR results in the qPCR device and helped to see if the method works as expected. In the final reaction, the EXPAR will be done in the absence of SYBR Green I, because the final quantification will be done by the toehold switches and thus it will not affect the final results. Nevertheless, more experiments are needed before this to standardize our method.

### **| Electrophoresis**

For the gel electrophoresis experiments, we used 4% agarose gel, because the DNA parts we wanted to separate were approximately between 30 and 80 bases [3]. Because of the very short length of the DNA fragments we wanted to visualize, we should have used the polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments [4]. Unfortunately, we lacked the necessary materials, equipment, and time. Thus, the electrophoresis results should be carefully examined.

![](https://static.igem.org/mediawiki/2021/e/e1/T--Thessaloniki--electro_1.png)
![](https://static.igem.org/mediawiki/2021/8/87/T--Thessaloniki--electro_2.png)

Figure 23: 4% agarose gel for the products of EXPAR – DNA Protocol at 65 ℃. Left (A): Shows the agarose gel after 20 minutes of electrophoresis at 88V. Right (B): Shows the agarose gel after 35 minutes of electrophoresis at 88V

As shown in Figure 23, in the products of EXPAR – DNA Protocol at 65 ℃, there are a lot of by-products, as the lower band of each well indicates. But this should not be a problem, because the final quantification will be done by the toehold switches which are very specific for the microRNAs and should not detect the by-products of the reaction. On the contrary, SYBR Green I is not specific and is binding to every dsDNA [5].

For the EXPAR – DNA Protocol at 37 ℃, in the gel electrophoresis, there were no bands in the wells that the products were loaded. During the gel preparation, due to the high agarose concentration (4%) and the fact that we didn’t use low melting temperature agarose, the lower wells were damaged and probably the samples weren’t loaded in the wells properly.

For the final EXPAR – DNA and RNA Protocol at 37 ℃, the gel electrophoresis results are shown in Figure 24, which confirms partially the amplification plot results. The amplification of the microRNAs is easily seen in Figure 24. c, in the form of the lower band of each well. In the negative controls, there were by-products, too. A polyacrylamide gel electrophoresis (PAGE) should be conducted for safer results.

![](https://static.igem.org/mediawiki/2021/7/7d/T--Thessaloniki--electro_30e_20.png)
![](https://static.igem.org/mediawiki/2021/a/ab/T--Thessaloniki--electro_30e_30.png)
![](https://static.igem.org/mediawiki/2021/7/7c/T--Thessaloniki--electro_1246.png)

Figure 24: 4% agarose gel for the products of Primer ligation with T4 Protocol

## References

* **|[1]** New England Biolabs, “T4 DNA Ligase”, Instruction Manual, 2021, https://international.neb.com/products/m0202-t4-dna-ligase#FAQs%20&%20Troubleshooting, (accessed Oct 16, 2021)
* **|[2]** Y. G. Ren, G. Martinez, L. A. Kirsebom, A. Virtanen, “Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides”, RNA, vol. 8, no. 11, pp. 1393–1400, Nov. 2002
* **|[3]** Thermo Fisher Scientific, “General Recommendations for DNA Electrophoresis”, protocol, 2012, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012614\_Gen\_Recommend\_DNA\_Electrophoresis\_UG.pdf, (accessed Oct 16, 2021)
* **|[4]** Addgene, “Agarose Gel Electrophoresis” protocol, https://www.addgene.org/protocols/gel-electrophoresis/ (accessed Oct 16, 2021)
* **|[5]** N. Marmiroli, E. Maestri, Chapter 6 - Polymerase chain reaction (PCR), Food Toxicants Analysis, Elsevier, pp. 147-187, Feb. 2007, ISBN 9780444528438

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