iGEM 2019 — Team Thessaly

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# Team:Thessaly/Notebook

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About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education & Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Thessaly--logo-mple.png)](https://2019.igem.org/Team%3AThessaly)

About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education &
 Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

☰

[Logo](https://2019.igem.org/Team%3AThessaly)

About

[Our Story](https://2019.igem.org/Team%3AThessaly/Our_Story)
[Members](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Gallery](https://2019.igem.org/Team%3AThessaly/Gallery)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Notebook](https://2019.igem.org/Team%3AThessaly/Notebook)
[Contribution](https://2019.igem.org/Team%3AThessaly/Contribution)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Improve](https://2019.igem.org/Team%3AThessaly/Improve)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)

Parts

[Parts Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Basic Parts](https://2019.igem.org/Team%3AThessaly/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AThessaly/Composite_Part)
[Parts Collection](https://2019.igem.org/Team%3AThessaly/Part_Collection)

Human Practices

[Education & Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Product Development](https://2019.igem.org/Team%3AThessaly/Product_Developmentct)

[Logo](https://2019.igem.org/Team%3AThessaly)

About

[Our Story](https://2019.igem.org/Team%3AThessaly/Our_Story)
[Members](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Gallery](https://2019.igem.org/Team%3AThessaly/Gallery)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Notebook](https://2019.igem.org/Team%3AThessaly/Notebook)
[Contribution](https://2019.igem.org/Team%3AThessaly/Contribution)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Improve](https://2019.igem.org/Team%3AThessaly/Improve)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)

Parts

[Parts Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Basic Parts](https://2019.igem.org/Team%3AThessaly/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AThessaly/Composite_Part)
[Parts Collection](https://2019.igem.org/Team%3AThessaly/Part_Collection)

Human Practices

[Education & Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Product Development](https://2019.igem.org/Team%3AThessaly/Product_Development)

☰

[TOP](#top)

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TITLE

[link 1](#11)
[link 2](#12)
[link 3](#13)
[link 4](#14)

**link 1**

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**link 2**

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**References**

[![](https://static.igem.org/mediawiki/2019/0/05/T--Thessaly--facebook.png)](https://www.facebook.com/igemthessaly/?epa=SEARCH_BOX)
[![](https://static.igem.org/mediawiki/2019/5/55/T--Thessaly--youtube.png)](https://www.youtube.com/channel/UCBHXzFL7r9xxHxqQICznphA)
[![](https://static.igem.org/mediawiki/2019/5/5a/T--Thessaly--twitter.png)](https://twitter.com/igemthessaly?lang=en)
[![](https://static.igem.org/mediawiki/2019/2/24/T--Thessaly--instagram.png)](https://www.instagram.com/igemthessaly/?hl=el)
[![](https://static.igem.org/mediawiki/2019/a/a1/T--Thessaly--search.png)](http://igem-thessaly.uth.gr/)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Thessaly/Experiments

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Thessaly--logo-mple.png)](https://2019.igem.org/Team%3AThessaly)

About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education & Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Thessaly--logo-mple.png)](https://2019.igem.org/Team%3AThessaly)

About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education &
 Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

☰

Sorry, your browser does not support inline SVG.

[![](https://static.igem.org/mediawiki/2019/7/7d/T--Thessaly--logo-projject.svg)](https://2019.igem.org/Team%3AThessaly)

EXPERIMENTS.

[Protocols](#11)
[Experimental Design](#12)
[- *IS6110* preparation](#121)
[- DNA Amplification](#122)
[- In vitro protein synthesis](#123)
[- Two steps into one](#124)
[- Universal Toehold switches](#125)
[- New disease](#126)
[Lab Book](#13)

[![](https://static.igem.org/mediawiki/2019/f/fe/T--Thessaly--up.png)](#top)

![](https://static.igem.org/mediawiki/2019/a/af/T--Thessaly--overview.png)

The workflow of our experiments was based mainly on the design of our project, which consists of 3 basic parts. The amplification of the biomarker sequence, followed by *in vitro* transcription and translation, resulting to a color-changing reaction. In this page you can find the protocols used during our experiments, our detailed experimental design, and the Lab Book of our project.

**Protocols**

For an experiment to be rightly executed, some basic protocols must be followed. Here is the list and the detailed description of the protocols we used, in order to get through the experiments. To download our protocols click in the boxes below.

[Chemically Competent Cells](https://static.igem.org/mediawiki/2019/d/df/T--Thessaly--ChemicallyCompetentCells.pdf)
[Inoue Chemically Competent Cells](https://static.igem.org/mediawiki/2019/7/7e/T--Thessaly--InoueChemicallyCompetentCells.pdf)
[Restriction Digest](https://static.igem.org/mediawiki/2019/e/e9/T--Thessaly--RestrictionDigest.pdf)
[DNA Ligation](https://static.igem.org/mediawiki/2019/7/7f/T--Thessaly--DNALigation.pdf)

[Transformation](https://static.igem.org/mediawiki/2019/a/aa/T--Thessaly--Transformation.pdf)
[Minipreps Plasmid Isolation](https://static.igem.org/mediawiki/2019/3/32/T--Thessaly--MiniprepsPlasmidIsolation.pdf)
[Glycerol Stock Preparation](https://static.igem.org/mediawiki/2019/e/ea/T--Thessaly--GlycerolStockPreparation.pdf)
[Primer Preparation](https://static.igem.org/mediawiki/2019/e/e2/T--Thessaly--PrimerPreparation.pdf)

[PCR Kapa Taq](https://static.igem.org/mediawiki/2019/1/18/T--Thessaly--PCRKapaTaq.pdf)
[PCR - Q5](https://static.igem.org/mediawiki/2019/3/3e/T--Thessaly--PCRQ5.pdf)
[Colony PCR](https://static.igem.org/mediawiki/2019/4/4a/T--Thessaly--ColonyPCR.pdf)
[PCR Cleanup](https://static.igem.org/mediawiki/2019/b/b7/T--Thessaly--PCRClearup.pdf)

[Gel Preparation and Run](https://static.igem.org/mediawiki/2019/5/53/T--Thessaly--GelPreparationAndRun.pdf)
[Gel Extraction](https://static.igem.org/mediawiki/2019/9/96/T--Thessaly--GelExtracton.pdf)
[PURExpress](https://static.igem.org/mediawiki/2019/c/c4/T--Thessaly--PURExpress.pdf)
[RPA](https://static.igem.org/mediawiki/2019/e/ef/T--Thessaly--RPA.pdf)

[DNase I Treatment](https://static.igem.org/mediawiki/2019/9/93/T--Thessaly--DNaseITreatment.pdf)
[*In vivo* Protein Expression](https://static.igem.org/mediawiki/2019/f/fa/T--Thessaly--InVivoProteinExpressionProtocol.pdf)
[TAE Solution](https://static.igem.org/mediawiki/2019/4/44/T--Thessaly--TAESolution.pdf)
[M9 medium](https://static.igem.org/mediawiki/2019/e/e9/T--Thessaly--M9Medium.pdf)

**Experimental Design**

***IS6110* preparation**

The first step of our experiments was to prepare our biomarker, the *IS6110* gene fragment. We first had to order the sequence of the *IS6110* fragment from IDT (Integrated DNA Technologies) and after diluting it to the desired concentration, to amplify it with a set of primers that anneal at the edge of our DNA fragment. This step was necessary in order to amplify our sequence and keep it stored so that it can be used at later steps as a template for our experiments. It is important to note that our fragment was ordered without the prefix and suffix sequences, thus, in order to make it RFC10 compatible, we added these sequences as overhangs in the abovementioned primers. After obtaining this sequence in many copies, we had to quantify it and later create a series of dilutions of our fragment so that they could be used later as a template to test the efficiency of our amplification method (RPA).

![](https://static.igem.org/mediawiki/2019/9/9a/T--Thessaly--exp1.png)

**Figure 1.** Vector map of pSB1C3 containing the IS6110 gene fragment (987bp)

**DNA Amplification using Recombinase Polymerase Amplification (RPA) and PCR**

Our first step was to check if our chosen biomarker (*IS6110* element) can be amplified, first with a PCR reaction and then with the Recombinase Polymerase Amplification (RPA), which is an isothermal amplification method. In order to determine the reaction’s optimal conditions for the selected DNA template, we conducted a series of experiments. Ultimately, we were able to show that the amplification of our DNA biomarker can be achieved using both PCR and RPA.[1]

Briefly, we examined the amplification performance both for the PCR and the RPA reaction when altering the following reaction conditions, one at a time, thus **standardizing the methods:**

**Standardization process**

**a. Amplified product length**

To test the reaction’s efficiency when amplifying different product sizes, we designed three sets of primers on our DNA template, corresponding to amplicon length of 80, 250 and 500 bp respectively. The reverse primer is the same in every set to ensure the similarity between the sets, in order to clarify if product length plays a role in amplification performance.[1]

**b. Annealing primer length**

Primer quality is a critical component of these reactions and especially for the RPA reaction. Even differences in a few nucleotides can increase the specificity of the reaction a lot. To address that, we designed three different reverse primers for our DNA template, each one with a different annealing length. Moreover, we sought to cover the range of optimal primer length according to RPA’s manual, thus the 3 oligos were 32, 36 and 39 bp in length respectively.

**c. Addition of 5’ end overhangs in the primer sequences**

In order to uncouple downstream signaling from the specific sequence of our DNA template, whilst creating a modular and universal tool, we designed primers with additional sequences at their 5’ end. With a few publications on this matter, we sought to test reaction performance in presence of universal overhanging sequences.

[RPA & PCR Primers](https://static.igem.org/mediawiki/2019/5/54/T--Thessaly--RPA%2BPCR_Primers_FULL.xlsx)

**Note:** To see a detailed description of our *in silico* primer design, click [**here**](/Team%3AThessaly/Model#14).

**d. Incorporation of PCR additives**

Even though RPA is a great method to amplify and detect DNA in a very fast and cheap way, some intrinsic issues lie within its molecular mechanism. This results in frequent primer-dimer formation and presence of several RPA artifacts, along with the amplified sequence. To eliminate those, we tested some common PCR additives that are known to enhance a PCR reaction’s specificity, both for the PCR and the RPA reactions. Moreover, since *Mycobacterium tuberculosis* and thus our biomarker has a high GC content, those additives can help alleviate secondary structures in the template. To be precise, we tested the PCR performance using **DMSO (Dimethyl Sulfoxide)**Molecular grade DMSO is a PCR additive which is commonly used for the amplification of GC rich templates. It enhances the reactions performance by facilitating the specific annealing of the primers to the template and thus, reducing non-specific amplification at a final concentration of 5% and the RPA performance, also with the addition of DMSO (2.5%, 5%, 10%). While DMSO improved the PCR performance significally, it seems to inhibit the reaction of RPA, and that is maybe the reason for the lack of literature on the matter[2].

To further standardize the RPA method, which is the amplification method we chose for our project design, we conducted some more experiments to check different conditions for our method and establish which of those are the optimal ones.

**e. Incubation temperature (25℃, 37℃, 39℃, 42℃, 50℃)**

Another major requirement is the conduction of the assay without using specialized equipment. Thus, heating in a specific temperature can decrease the overall impact of the test. RPA has a recommended incubation temperature of 39℃ , so in order to explore the limits of that, we tested reactions at **25 (RT), 37℃, 39℃, 42℃ and 50℃**, showing that while the optimal range is between 37-42℃, the reaction can be conducted in either higher or lower temperatures.

**f. Template Concentration**

One of the most critical aspects of a diagnostic test is the assay’s sensitivity and thus the Limit of Detection (LoD). In order to assess the LoD of RPA with our biomarker, we prepared serial dilutions of the template, down to 1 copy per μl (corresponding 10-9ng/μl). Furthermore, we conducted RPA reactions successfully with **1, 10, 102 , 103, 104, 105, 106, 107, 108 copies** of our biomarker **per reaction** respectively.

**g. Incubation time (3, 5, 7, 10, 15, 20 min)**

A requirement of Point of Care (POC) tests is that they can produce a reliable signal in-field, in a small fraction of time. Thus, we assessed RPA performance in decreasing incubation time. Furthermore, starting from the recommended incubation time of 20 minutes, we decreased it to 15, 10, 7, 5 and 3 minutes respectively, managing to amplify even a single copy in a 5-minute reaction.

**Optimal conditions in RPA for our implementation**

Combining the knowledge gained from the standardization process, we performed the final optimized RPA reactions for our MTB biomarker, in a modified protocol where incubation time is 5 minutes, the temperature is 42℃, while being able to detect even a single copy of the template per reaction.

**DNA fragmentation**

To further enhance our proof of concept and make the conditions of our experiments seem more realistic, we decided to test our diagnostic test, simulating the natural fragmentation of our biomarker (*IS6110*) in patients’ urine and see if we would be able to locate and amplify it. For this reason, we used DNase I to our template DNA (987bp), so that random fragmentation would occur, and after that, we were able to still amplify our biomarker with our designed primer set and in much more realistic conditions.[4,5]

***In vitro* protein synthesis assay**

The second experimental installment of our project was the expression of the protein of interest, through a cell-free system. The goal of this experiment was to prove that the device we have designed for DNA detection can produce a robust colorimetric readout, both visible to the naked eye and measured in a plate-reader spectrophotometer. To achieve that, we conducted several preliminary experiments, to prepare and calibrate the reaction’s conditions, as well as produce the genetic material used in the reactions.

The steps we followed towards our goal are:

**Cloning of the genetic material**

In order to have enough genetic material for all our experiments, we spared some time to clone every BioBrick we received into pSB1K3, pSB1C3 of pSB1A3 clining vectors. Below you can find a table with the BioBricks cloned and used in our *in vitro* experiments (all parts have a T7 promoter [**BBa\_J64997**](http://parts.igem.org/Part%3ABBa_J64997) and T7 terminator [**BBa\_B0015**](http://parts.igem.org/Part%3ABBa_B0015)).

**Table 1.** The BioBrick parts that were generated and used in our *in vitro* protein synthesis assay

**BioBrick Name**

**Cloned in**

**Selection with**

**Registry Parts**

**eGFP**

pSB1C3, pSB1K3

Chloramphenicol, Kanamycin

[**BBa\_K2973002**](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2973002)

**Toehold-switch regulated eGFP**

pSB1C3, pSB1K3

Chloramphenicol, Kanamycin

[**BBa\_K2973006**](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2973006)

**β-lactamase**

pSB1A3

Ampicillin

[**BBa\_K2973000**](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2973000)

**Toehold-switch regulated
β-lactamase**

pSB1C3

Chloramphenicol

[**BBa\_K2973007**](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2973007)

**Trigger sequence**

pSB1A3

Ampicillin

The non-regulated parts serve as a measure to compare the regulation efficiency of the toehold switch.

After the cloning process, we isolated the plasmid containing each part in large concentrations and confirmed their identity via Sanger Sequencing. At that point we had enough genetic material to use in our *in vitro* experiments downstream.

**Calibrating the cell-free protein expression reaction**

The next step was to test and calibrate the *in vitro* protein synthesis reaction, due to the lack of expertise in such reactions here in our Department. We used the commercial kit PURExpress from NEB (New England Biolabs) to express the proteins of interest *in vitro*. Even though there is a detailed protocol available, we optimized its parameters to suit our needs. Moreover, we compared protein expression through an enzymatic assay, between the regulated and non-regulated genes. The conditions we altered are:

a. The volume of the reaction reduced to 7 ul from 25 ul

b. Incubation time (1, 2 and 3 hours)

c. Abundance of trigger sequence in the reaction (75, 50, 16, 7, 3, 0.3 nM per reaction)

d. Different reporter genes (eGFP, β-lactamase)

Our reporter gene of interest is β-lactamase, but during the calibration process we tested eGFP as well, which is a robust reporter protein and can be easily detected, both *in vivo* and *in vitro*. As a result, we have provided the community with two evidently working BioBricks regulated by an orthogonal toehold switch.

**Note:** We ensured that β-lactamase's activity is due to the regulated gene only, because PURExpress utilizes only T7 promoters to transcribe and translate coding sequences.

**Performing the proof of concept reactions**

In order to prove that β-lactamase can be expressed in regulation of an orthogonal switch, we performed a batch of reactions. These included both positive (unregulated gene) and negative (regulated gene without the trigger sequence) controls. After the addition of the substrate in each reaction, we performed an established enzymatic assay for the β-lactamase enzyme for 30 minutes.

**Combining the two separate proof of concepts into one**

After completing the proof of concept for the toehold switch, we wanted to test the complete functionality of our project and prove that these uncoupled and separate reactions (RPA and toehold switch regulated expression on β-lactamase) can perform as a combined reaction. To test this, we had first to perform the RPA reaction as usual and then use the amplified product as the trigger sequence expressed with the PURE express *in vitro* synthesis kit, which will trigger our toehold switch to open its formation and enable for the β-lactamase enzyme to be expressed and give us a colorimetric (red) signal through the hydrolysis of Nitrocefin.

The workflow was based on the separate proof of concepts and the standardization of the techniques done previously. The RPA reaction was a 5 minutes reaction, while the product was cleaned after the amplification. Afterward, the amplified and cleaned product was used as a template for the PURE *in vitro* synthesis reaction which was a 3 hours reaction and then a 30-minute enzymatic assay followed to see if our amplified product can regulate our toehold switch. As a control we used both the β-lactamase enzyme not regulated by any toehold switch and the toehold itself without the triggering sequence, to check the levels of its leakage.

We concluded that with the right conditions, our template biomarker (*IS6110*) can be amplified by the RPA reaction and then, after its *in vitro* expression, can give the signal to regulate our toehold switch and lead to the expression of the β-lactamase enzyme and, thus, the hydrolysis of Nitrocefin and a vibrant color change. In conclusion, we showed that our proof of concepts can work together as one and give a universal platform for our diagnostic tool.

**Testing our new universal toehold switch**

Moving forward from the basic proof of concept, we de novo [**designed**](https://2019.igem.org/Team%3AThessaly/Model#14) a pool of new toehold switches to create a universal tool. After the *in silico* analysis two of them were selected to be tested. To prove that the freshly designed switches work, we performed a batch of reactions, following the proof of concept reaction conditions. Briefly, 7μl reactions were performed for 3hours *in vitro* transcription/translation and the nitrocefin substrate was additionally added. The toehold switch concentration was always the same while the trigger’s concentration was the one changing. We showed that one of the new toehold switches can indeed regulate the protein expression of β-lactamase and produce a robust signal.

![](https://static.igem.org/mediawiki/2019/e/ec/T--Thessaly--exp2.png)

**Figure 2.** Vector maps of pSB1C3 containing the trigger sequence (left) and toehold sequence (right) deriving from the Geobacillus kaustophilus

**New disease (*Hepatitis B*  Virus)**

With our design we aimed to develop a platform/tool that its nature of approach is universal, and simply by changing the primer set, **we can be able to target different pathologic agents**, as well as **other MTB traits, such as antibiotic resistance**. For us to be able to prove the universality of our design we had to test its efficiency when targeting something different. We chose to target a small DNA sequence of the *Hepatitis B* Virus (HBV), thus proving that with our design, it is possible to diagnose any other disease (if targeting its DNA) only by changing the primers’ sequences. We began exploring this different approach by choosing the targeted region. The region we chose is a small fragment of the DNA sequence of HBV (280 bp) that is part of 3 alternate reading frames. Then we had to redesign our already existing primers, changing only the annealing sequences and keeping the overhanging sequences stable. We designed 3 primer sets, based on pre-existing literature while adjusting their length to fit the RPA method standards.[6]

The first step of our experiments was to order the DNA fragment for HBV as well as the designed primers. After that, when the DNA fragment and the primers arrived, we had to create again a series of dilutions so that they could be used as a template to test the efficiency of our amplification method.

Our goal was to test all 3 primer sets, to check if they can amplify our “biomarker” through PCR and Recombinase Polymerase Amplification (RPA). At the same time, we wanted to check which one of these 3 sets amplifies our fragment most efficiently.

We started testing our primer sets first through PCR, using all 3 sets, while using as a template the lowest concentrations we had created. The conditions were the same as those used for our proof-of-concept reactions, while we used DMSO at a final concentration of 5% in all of our PCR reactions.[7,8]

After achieving the amplification with all of the aforementioned primers, and while having a small clue that the 1st set is able to amplify our fragment best, we continued by testing the amplification efficiency with the RPA method, again with the lowest concentrations of DNA template. The conditions we chose for the RPA reactions were exactly the same as used for our proof of concept reactions, which means that the incubation time was 5 minutes while the temperature was 42℃.

Again, we were able to show that the amplification of our DNA biomarker (this time from HBV) can be achieved using both PCR and RPA, and thus proving that we can use our initial design, with small modifications, to target different DNA templates, making it a universal and modular tool, and simply by changing the primer set, we can be able to target different pathologic agents and more.

**Lab Book**

One of the most crucial step of the lab work, is to report the daily conducted experiments in detail. Below is our team’s lab book describing our experimental routine. To download it click below.

[Lab Days](https://static.igem.org/mediawiki/2019/8/8b/T--Thessaly--LabDays.pdf)

**References**

**1.** TwistAmp® DNA Amplification Kits, Assay Design Manual

**2.** https://www.sigmaaldrich.com/catalog/product/sigma/d8418?lang=en&region=GR

**3.**L. Lillis, et al., Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care, *Molecular and Cellular Probes* (2016) ” ,*Infection*. https://doi.org/10.1007/s15010-016-0955-2.

**4.** Labugger, Ines, Jan Heyckendorf, Stefan Dees, Emilia Häussinger, Christian Herzmann, Thomas A Kohl, Elvira Richter, Eric Rivera Milla, and Christoph Lange. 2016. “Detection of Transrenal DNA for the Diagnosis of Pulmonary Tuberculosis and Treatment Monitoring.

**5.** Karayiannis, P, D M Novick, A S F Lok, and H C Thomas. 1985. “Hepatitis B Virus DNA in Saliva , Urine , and Seminal Fluid of Carriers of Hepatitis B e Antigen” 290 (June): 1853–55.

**6.**  Samejima, K., & Earnshaw, W. C. (2005). Trashing the genome: the role of nucleases during apoptosis. *Nature Reviews Molecular Cell Biology, 6*(9), 677–688. doi:10.1038/nrm1715

**7.** Alves, Danielle, Gomes Zauli, Carla Lisandre, Paula De Menezes, Cristiane Lommez, De Oliveira, Elvis Cristian, Cueva Mateo, Alessandro Clayton, and De Souza Ferreira. 2016. “Genetics and Molecular Microbiology In-House Quantitative Real-Time PCR for the Diagnosis of Hepatitis B Virus and Hepatitis C Virus Infections.” *Brazilian Journal of Microbiology 47* (4): 987–92. https://doi.org/10.1016/j.bjm.2016.07.008.

**8.**Jain, Surbhi, Ying-hsiu Su, Yih-ping Su, Sierra Mccloud, Ruixia Xue, Tai-jung Lee, Shu-chuan Lin, et al. 2018. “Characterization of the Hepatitis B Virus DNA Detected in Urine of Chronic Hepatitis B Patients,” 19–21.

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# Team:Thessaly/Results

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Thessaly--logo-mple.png)](https://2019.igem.org/Team%3AThessaly)

About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education & Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

[![](https://static.igem.org/mediawiki/2019/b/b1/T--Thessaly--logo-mple.png)](https://2019.igem.org/Team%3AThessaly)

About

[Our journey](https://2019.igem.org/Team%3AThessaly/Our_Journey)
[Team](https://2019.igem.org/Team%3AThessaly/Team)
[Attributions](https://2019.igem.org/Team%3AThessaly/Attributions)
[Collaborations](https://2019.igem.org/Team%3AThessaly/Collaborations)

Project

[Description](https://2019.igem.org/Team%3AThessaly/Description)
[Design](https://2019.igem.org/Team%3AThessaly/Design)
[Experiments](https://2019.igem.org/Team%3AThessaly/Experiments)
[Results](https://2019.igem.org/Team%3AThessaly/Results)
[Demonstrate](https://2019.igem.org/Team%3AThessaly/Demonstrate)
[Dry Lab](https://2019.igem.org/Team%3AThessaly/Model)
[Safety](https://2019.igem.org/Team%3AThessaly/Safety)
[Graphic Design](https://2019.igem.org/Team%3AThessaly/Graphic_Design)

Parts

[Overview](https://2019.igem.org/Team%3AThessaly/Parts)
[Characterization](https://2019.igem.org/Team%3AThessaly/Characterization)
[Improvement](https://2019.igem.org/Team%3AThessaly/Improve)

Human Practices

[Education &
 Public Engagement](https://2019.igem.org/Team%3AThessaly/Public_Engagement)
[Integrated](https://2019.igem.org/Team%3AThessaly/Human_Practices)
[Healthcare Model](https://2019.igem.org/Team%3AThessaly/Healthcare_Model)

Product Development

[Product Design](https://2019.igem.org/Team%3AThessaly/Applied_Design)
[Entrepreneurship](https://2019.igem.org/Team%3AThessaly/Entrepreneurship)

[Medal Criteria](https://2019.igem.org/Team%3AThessaly/Medal_Criteria)
[Giant Jamboree](https://2019.igem.org/Team%3AThessaly/Giant_Jamboree)

☰

Sorry, your browser does not support inline SVG.

[![](https://static.igem.org/mediawiki/2019/7/7d/T--Thessaly--logo-projject.svg)](https://2019.igem.org/Team%3AThessaly)

RESULTS.

[DNA amplification](#11)
[-PCR](#111)
[-RPA](#112)
[-Universality](#113)
[-Realistic conditions](#115)
[-Outlook](#114)
[*In vitro* protein synthesis](#12)
[-Calibration](#121)
[-Testing β-lactamase](#122)
[-Amplicon as the trigger](#123)
[-Universal toehold switches](#124)
[Future vision](#13)

[![](https://static.igem.org/mediawiki/2019/f/fe/T--Thessaly--up.png)](#top)

![](https://static.igem.org/mediawiki/2019/a/af/T--Thessaly--overview.png)

We conducted experiments to validate our designed primer sets amplification performance both in standard PCR and in the RPA reaction. After some rounds of optimization, we were able to amplify our biomarker (*IS6110*) in both types of reaction. Subsequently, we tested the functionality of our reporter system, initially using a well-characterized toehold switch from the literature and then with a newly designed toehold.

**DNA amplification**

We first tested our designed primers in PCR reactions, as this is the standard amplification method and thus more likely to succeed. Our goal was to choose the correct reaction conditions (primer combinations, PCR additives, *etc.*) to be used subsequently in RPA reactions.

**PCR**

**Testing our overhangs**

Our experimental design requires addition of overhanging sequences to the 5’ ends of our primers. We tested whether the addition of these overhangs affects the capacity of our designed primer sets to amplify the chosen biomarker sequence. As depicted in Figure 1, the amplification was successful, both with and without overhangs. Primers with overhangs yielded a 136bp sequence, while primers without overhangs resulted in a 80bp sequence.

![](https://static.igem.org/mediawiki/2019/f/f8/T--Thessaly--Results_Figure1.jpg)

**Figure 1.** PCR amplification of our DNA biomarker both with and without the 5’ universal overhanging sequences we designed.

**Primer screening**

After proving that primers with 5’ overhangs can amplify our biomarker in a PCR reaction, we needed to validate a number of primer pairs for optimal functionality. We screened a total of 9 primer combinations, which yield different amplified product lengths (136, 306 and 556 bp) and different annealing lengths for the reverse primer (32, 36 and 39 bp annealing to the template). With this reaction we sought to test those different primer combinations and their probing efficiencies. This is crucial, because according to the literature and our project design, the biomarker DNA templates we aim to amplify are generally 150 bp long[1].

As depicted in figures 2A and 2B, all primer combinations tested can amplify the target biomarker. Combinations F1-R1, F1-R2 and F1-R3 give 136bp amplicon, while the F2 series gives the 306bp and the F3 the 556bp, respectively. Based on these results and because a short amplicon was desirable, we chose the first forward primer(F1) depicted above with the second corresponding reverse primer (R2), which according to our *in silico* analysis is the best of the three reverse primers.

![](https://static.igem.org/mediawiki/2019/b/b8/T--Thessaly--Results_Figure2AB.png)

**Figure 2. A)** Primer screening of 3 primer combinations with the 1st Forward primer, which correspond to ~136bp amplified product lengths and different annealing lengths for the reverse primers (32, 36 and 39 bp annealing). **B)** Primer screening of 6 primer combinations with the 2nd and 3rd Forward primer, which correspond to ~306 and 556bp amplified product lengths and different annealing lengths for the reverse primers (32, 36 and 39 bp annealing).

**Sensitivity test and incorporation of PCR additives**

Having chosen the best primer pair to be used in our proof of concept reactions, we needed to further validate its suitability and sensitivity of detection at a range of template mimicking biologically relevant levels of the target biomarker. To this end, reactions took place using two different template concentrations, resembling to 1 and 10 copies per reaction, respectively. In Figure 3A, we can clearly see that the selected primers can produce a specific band of substantial intensity, even when 1 copy of the biomarker sequence is present. Concurrently, we checked our reaction’s performance after the addition of a PCR additive, DMSO, as it is known to enhance the efficiency of amplification reactions (Fig. 3B). Indeed, we were able to correctly amplify our biomarker in both reactions, while proving that we can amplify as much as a single copy of our biomarker with our designed primers.

![](https://static.igem.org/mediawiki/2019/0/0a/T--Thessaly--Results_Figure3AB.png)

**Figure 3. A)** PCR amplification of our DNA biomarker *IS6110* in the lowest concentrations of our DNA template. **B)** PCR amplification of our DNA biomarker *IS6110* after the addition of DMSO at a final concentration of 5%.

**RPA**

Having a validated primer pair and identified a range of suitable reaction conditions by PCR, we continued with our main objective. That is, testing whether RPA is capable of giving similar results to PCR and of detecting the target biomarker in conditions similar to those that might be found in biological samples.

We examined a range of different reaction conditions, including those tested previously by PCR reactions in order to overcome technical issues with RPA, including non-specific amplification in our reactions. An example of our initial RPA reactions is shown in Figure 4. Even though RPA is optimized to work for amplicons up to 500bp, we were unsuccessful when trying to amplify the 500bp amplicon primer set. Moreover, when no template was added, non-specific amplification occurred. We hypothesize that this is caused due to the formation of secondary structures of the primers, since their length lies within the 60-80bp range.

![](https://static.igem.org/mediawiki/2019/b/b7/T--Thessaly--Results_Figure_4.png)

**Figure 4.** RPA test reactions for some primer combinations.

**Testing different reaction conditions**

Even though RPA is a great method to amplify and detect DNA in a very fast and relatively cheap way, according to the literature[2], its molecular mechanism comes with some intrinsic shortcomings that give rise to potential problems. More specifically, because an RPA reaction is carried out well below the minimum annealing temperature of all dsDNA molecules, this results in frequent primer-dimer formation and presence of several RPA artifacts, along with the desired amplified sequence. Thus, we concluded that it is possible that our chosen primers, due to their length and perhaps additional physicochemical characteristics attributed to their specific sequence, form primer-dimer formations. This in turn, leads to one or more non-specific products being visualized both in test reactions and in negative no-template control reactions. Wanting to further test our reactions under different conditions and check if this will help alleviate the possible secondary structures that may form in our reaction, we tested different reaction times and different temperatures for the PRA with our chosen primers.

Although still we could see an amplified product in the NTCs, we validated that the RPA reaction can amplify our biomarker even in a 5-minutes reaction (Fig. 5A). The standard RPA protocol includes a 20 minute reaction, while there are reported successful incubation times of 3 minutes. We tested a range of incubation times within these limits. As seen in Figure 5A, amplification can be witnessed until the 5min lane on the agarose gel. Moreover, as seen in Figure 5B the reaction shows higher efficiency when the incubation temperature is at 42˚C. This is logical, since the kinetics of the reaction are proportional to temperature.

![](https://static.igem.org/mediawiki/2019/2/24/T--Thessaly--Results_Figure5A%CE%92.png)

**Figure 5. A)** RPA in different reaction times. **B)** RPA in different reaction temperatures.

**Chosen conditions**

In order to overcome these issues, we continued our tests so that we can achieve the optimal conditions for our reaction, without any non-specific amplification. Our next step was to combine the 5-minute reaction time and the 42˚C reaction temperature, based on the rationale that the shorter reaction time in combination with a higher temperature could alleviate the possible secondary structures of our reaction. This took into account our *in silico* analysis, which predicted that possible secondary structures should not appear above 42˚C. At the same reactions we also tested serial dilutions of our template DNA, to establish the sensitivity of the reaction.

This combination of changes to our approach resulted in the successful amplification of our target biomarker without any visible non-specific products in the negative control reaction (Fig. 6). We were also able to establish that the RPA reaction is able to amplify as little as a single copy of our biomarker with our designed primers, just like our PCR-based results, suggesting that our method is very promising in terms of sensitivity.

![](https://static.igem.org/mediawiki/2019/0/0e/T--Thessaly--Results_Figure_6.png)

**Figure 6.** RPA reaction for the detection of *IS6110* with serial dilutions of our DNA template, in a 5 min and 42 degrees Celsius reaction.

After successfully amplifying our target DNA through an RPA reaction, we needed to make sure that our successful result is reproducible. We performed another amplification reaction with the same conditions (Figure 7). This time we used a denser agarose gel (4%) with a low melt agarose (3:1), so we would be able to visualize any small differences in the sized of our amplified products.

As shown in Figure 7, we managed to correctly amplify our biomarker. Evidently, the 4% agarose gel allowed the fine separation of 2 different bands, which are around 10bp apart. We confirmed that the upper band is the specific product by PCR. It is thus clear that the detection of *IS6110* is successful, even if 1 template copy is used per reaction. Also, according to existing literature it is documented that (RPA) is prompt to give non-specific products in low template concentrations[2]. This is confirmed, since higher-copy reactions tend to diminish the intensity of the non-specific band. We hypothesize that when there is a high copy-number of DNA template in the reaction, less primers are available to form structures that allow non-specific amplification.

![](https://static.igem.org/mediawiki/2019/9/93/T--Thessaly--Results_Figure_7.png)

**Figure 7.** Amplification reaction with serial dilutions of our DNA template

**Testing the universality**

To test the universality of our design we tested 3 new sets of primers, in which we only changed the annealing sequence of the primer, to be able to aim for the chosen DNA sequence of the HBV virus, while retaining the appropriate 5’ overhangs necessary to trigger our downstream detection module. We started testing our primer sets first through PCR, using all 3 sets, while using as a template the lowest concentrations we had created. The conditions were the same as those used for our proof-of-concept reactions, while we used DMSO at a final concentration of 5% in all of our PCR reactions.

![](https://static.igem.org/mediawiki/2019/b/be/T--Thessaly--Results_Figure_8.png)

**Figure 8.** Primer screening for HBV.

After successfully amplifying our second biomarker through PCR (Fig. 8), we established that the best designed primer set was the first one, thus it was used on our next amplification reaction which was an RPA reaction with the same conditions tested for our proof of concept reactions with our first biomarker (*IS6110*).

![](https://static.igem.org/mediawiki/2019/c/cc/T--Thessaly--Results_Figure_9.png)

**Figure 9.** RPA reaction for the detection of HBV in the lowest concentration of our template DNA.

According to the results depicted in Figure 8 and 9, we concluded that we were able to amplify our chosen biomarker for the HBV virus, both with a PCR and an RPA reaction. We also attested that the amplification of this template DNA was more successful than that of the *IS6110* , which further enhances our hypothesis that the high GC% content of the *IS6110* gene is one of the main factors causing non-specific amplification. Still, RPA artifacts are present in the HBV reactions as well. However, since RPA artifacts are common and acknowledged in the literature and result in different lengths than the specific sequence, we hypothesize that they will not be an issue in downstream detection. This can be confirmed by sequencing the artifacts.

**Simulating realistic conditions**

After a series of exhaustive testing to optimize the amplification reaction, we still had not answered a vital question: Could we detect the biomarker in realistic conditions? To address that, we wanted to simulate the random fragmentation of the MTB genome when found in urine, so a fragmentation protocol was conducted. To ensure the non-specific manner and to simulate the conditions of the human body, a non-specific human endonuclease, DNAse I, was used. After a small series of calibrations to the DNAse I reaction, we managed to fragment the biomarker properly, as seen in Fig. 10.

![](https://static.igem.org/mediawiki/2019/6/67/T--Thessaly--Results_Figure_10.png)

**Figure 10.** DNase I fragmentation of *IS6110* biomarker for MTB detection. 3min and 6min correspond to the incubation time of the template with the enzyme at 37℃.

The next step was to attempt an amplification reaction on the randomly fragmented template. We successfully amplified the desired sequence from different fragmentation conditions, as depicted in Fig. 12.**This achievement holds great value, as we prove that our system would potentially work in clinical samples, where DNA is fragmented through a similar mechanism**.

![](https://static.igem.org/mediawiki/2019/e/ea/T--Thessaly--Results_Figure_11.png)

**Figure 11.** Amplification of randomly fragmented *IS6110* biomarker successfully using PCR.

**Outlook**

Although we were able to amplify target biomarkers for two different diseases using PCR and more importantly RPA, some issues still remain with regard to RPA specificity and fidelity. Some future plans for us to test, include be the incorporation of primers with SAMRS nucleotides at the 3’ end. According to the literature[3], this is a promising strategy to eliminate non-specific amplification. This plan was initially part of our experimental design, however, the cost for the purchase of these primer sets was too high for an iGEM team. However, our results show that in principle we can amplify target DNA at very low concentration and from shredded low MW templated DNA.

***In Vitro* protein synthesis assay**

In parallel with optimizing RPA-based target amplification, we began working towards the *in vitro* protein synthesis assay necessary for our detection system to function. For our experiments, we used the commercial PURExpress kit from New England Biolabs (NEB) , which is highly purified and reliable for a proof of concept experiment. The highlights of these experimental results are described below.

**Cloning Of The Genetic Material**

The first preliminary step towards testing our [**constructs**](https://2019.igem.org/Team%3AThessaly/Experiments#123), was to clone them into appropriate vectors for propagation. To do this, we cloned all necessary constructs in either pSB1K3, pSB1C3 of pSB1A3. As seen in Fig. 12, 13 most of the constructs are 900-1100bp, and thus appear around the 1000bp ladder marker. The trigger construct was the smallest and appears between the 100 and 200bp marker. The 2000bp band is the cut plasmid after the diagnostic digestion reaction. In figure 13, the image showing the cloning of β-lactamase represents a colony PCR reaction.

![](https://static.igem.org/mediawiki/2019/e/e5/T--Thessaly--Results_Figure_14.png)

**Figure 12.** 32B trigger cloning. C=Cut plasmid, U=Uncut plasmid.

![](https://static.igem.org/mediawiki/2019/a/ae/T--Thessaly--Results_Figure_12131516.png)

**Figure 13.** Cloning of 32BeGFP, eGFP, 32B β-Lactamase and β-Lactamase.

**Calibrating the cell-free protein expression system**

We calibrated our cell free system using of the genetic constructs described above. The aim was to initially examine the functionality of the cell-free system using non-regulated constructs and compare them with the results of the toehold-switch regulated constructs.

To do so, we incorporated two reporter proteins, eGFP and β-lactamase. β-lactamase is our reporter of choice for our current project, while eGFP is a common and well-characterized protein for easy visualization and quantification of performance that can be employed in other systems using our developed parts.

The first assay conducted was with eGFP as the reporter. To enable translation of the protein from the toehold regulated construct, different amounts of trigger sequence were added in the reaction. a reaction without a trigger sequence was also included as a negative control and a measure of background un-induced promoter activity. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL.

After a 3-hour incubation at 37℃ in a thermocycler, protein expression was measured using a plate-reader. To measure eGFP, excitation at 488nm was used, while detection was at 515nm, the protein’s maximum fluorescence emission point. The results of the assay can be seen at the graph below:

![](https://static.igem.org/mediawiki/2019/9/93/T--Thessaly--Results_Figure17.jpg)

**Figure 14.** eGFP fluorescence after *in vitro* protein expression

As expected, the non-regulated eGFP emitted the highest fluorescence, providing a positive control and confirming the protocol we used was functional. The toehold-regulated construct produced robust signal in decreasing concentrations of trigger (75nM, 15nM, 7nM). It is interesting that the signal remained the same even with a 10-fold decrease in trigger concentration, while the no trigger control had an almost 2-fold lower fluorescence. This indicates that there was strict regulation in the eGFP construct and higher concentrations of trigger sequences are needed for maximum activation.

**Testing the system with β-lactamase**

After ensuring the functionality of the system, we proceeded to test the β-lactamase constructs.

Following the same outline as before, we tested both a non-regulated construct, as well as a range of trigger concentrations and a background negative control. Since β-lactamase is an enzyme, it requires an enzymatic assay for the protein expression to be measured. To do this, after the 3-hour incubation in the cell-free system, we added a chromogenic substrate of β-lactamase, nitrocefin, and performed an additional enzymatic assay in a plate reader, at 37℃. Nitrocefin is yellow (with a maximum absorbance at 380nm) when not hydrolyzed, but upon hydrolysis by β-lactamase it converts to a red color (with a shift in maximum absorbance at 480nm). This provides a visual measure of β-lactamase activity that can also be quantified.

The results of the enzymatic assay are depicted in Fig. 15:

![](https://static.igem.org/mediawiki/2019/5/5a/T--Thessaly--Results_Figure18.jpg)

**Figure 15.** Enzymatic assay of β-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold regulated construct in a cell-free system for 3 hours. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted.

The positive control Lactamase ensures that the reaction was functional. Unlike eGFP, the 75nM trigger reaction produced a similar signal to the positive control reaction, which is promising. However, when lowering the trigger concentration to 7nM, the signal was almost halved. This is interesting, since both eGFP and β-lactamase were regulated by the same toehold switch. This might imply that the reporter gene and its nature (e.g. enzyme) can affect the produced signal. The no trigger control was substantially lower this time, showing an almost 4-fold decrease in comparison with the 75nM trigger reaction.

The next goal was to assess the performance of the enzymatic assay, when lowering the trigger concentrations and the incubation time. First, we decreased the trigger concentration even further, up to 0.3nM. When incubating for 3 hours the PURExpress reaction, even the 0.3nM reaction produced ~2-fold more signal, compared to the no trigger control (Fig. 16).

![](https://static.igem.org/mediawiki/2019/8/89/T--Thessaly--in_vitro_%CE%B2-Lactamase_3h.png)

**Figure 16.** *In vitro* transcription/translation of toehold-regulated β-lactamase with low concentrations of trigger sequence and quantification through a chromogenic enzymatic assay. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted.

This is promising, since the system can produce detectable signal with very low concentrations of trigger, thus increasing the sensitivity of our diagnostic test.

Since PURExpress’ incubation time is the most time-consuming part of our project, we intended to lower the incubation time and assess the performance of the cell-free system and its sensitivity. We performed 1-hour *in vitro* protein synthesis, with all the other conditions remaining the same. Again, a gradient of trigger concentrations was included to measure the system’s sensitivity. The results are shown in the figure (Fig.17):

![](https://static.igem.org/mediawiki/2019/9/9b/T--Thessaly--Results_Figure20.jpg)

**Figure 17.** 1-hour *in vitro* transcription/translation of toehold-regulated β-lactamase and quantification through a chromogenic enzymatic assay. Blank was subtracted.

To our surprise, the 75 nM trigger performed similarly to the 3-hour assay, while the 50 nM produced very similar signal. This might imply that those trigger concentrations are more than enough for reliable protein expression, since a 3-fold decrease in incubation time did not affect the signal, which reached a level comparable to the positive control. Also, both 15nM and 7nM trigger concentrations produced similar results to the 3-hour assay. However, the 3nM trigger concentration did not produce a strong signal, since it was not distinguishable from the no trigger control.

This implies that 7nM is the minimum trigger concentration per reaction, in 1-hour *in vitro* protein synthesis. This translates to ~500 picograms of trigger concentration DNA per reaction. In addition, a DNA amplification with PCR or RPA, produces 50-100nM of the engineered trigger sequence when 10-100 copies of initial DNA template is present respectively, as measured by our amplification team. Hence, it is safe to assume that a 1-hour *in vitro* protein synthesis reaction would produce a robust signal and provide reliable detection of the initial DNA template in a biological sample.

**Using RPA-amplified sequence as the trigger**

The goal of our project is to amplify – and at the same time engineer – a DNA sequence which acts as a trigger sequence for our toehold switch. Since both the amplification and *in vitro* protein synthesis steps were achieved, we sought to integrate them into one final experiment. In this setup, instead of expressing the trigger from a plasmid, we implemented the trigger sequence in the form of amplification product. In this way, we ultimately simulate the functionality of the system since the trigger sequence is linear, as it occurs after the amplification reaction.

In the final experiment, we followed the same working protocol in a 7μL reaction for *in vitro* protein synthesis of β-lactamase. The only key difference here, is that during the enzymatic assay we increased substrate concentration. This was done due to technical reasons, as in some previous experiments with low reaction volume (~50ul) the measurements from the plate-reader were sometimes inconsistent. To overcome this issue, we decided to increase the volume of the enzymatic assay to 100 ul, which resulted in much more consistent readings. Also, because of technical difficulties on purifying the amplicon from PCR/RPA in sufficient amounts, we added a synthesized sequence as a proof of concept, which is identical to the expected sequence from the amplification reaction.

After a 3-hour incubation in PURExpress, β-lactamase expression was measured in a 30-minute enzymatic assay. The results are depicted in Figure 18 below.

![](https://static.igem.org/mediawiki/2019/a/aa/T--Thessaly--Results_Figure_18a.png)

**Figure 18.** Measurement of toehold-regulated β-lactamase when the trigger is the previous step’s amplified sequence or in a plasmid vector. Error bars represent the standard deviation of n=2 replicates. Blank was substracted.

As seen in the graph, the produced signal from both trigger sources is closely similar, while a 5-fold increase is observed when compared to the no trigger control. We assume that the slight difference in concentration between linear and circular trigger is insignificant, since as shown before the system is saturated and fully activated when more than 50nM of trigger sequence are added.

After proving that both detection modules can work together, resembling a highly integrated system, we wanted to address one more thing. The toehold switch we had used so far was an established functional switch, while its trigger sequence derived from a Zika Virus strain (Toehold & trigger 32B,[4]). To avoid cross-talk and false positive results in the event of testing someone having Zika, we sought to address this issue by creating synthetic universal toehold switches.

**Universal toehold switches**

A key aspect of our project is to provide a tool able to detect the DNA of any organism easily and reliably. To this end, we *in silico* [**designed**](https://2019.igem.org/Team%3AThessaly/Model#112) a pool of 15 universal toehold switches from hyperthermophile bacteria. Due to budget limitations, only the two most promising switches were ordered and tested according to the *in silico* analysis, Toehold 13 and Toehold 14. Both toeholds had similar thermodynamics and thus we expected similar performance in terms of protein expression regulation.

To assess the new toeholds’ performance, we performed a series of *in vitro* protein synthesis reactions, following the same protocol and conditions as before. The major issue with toehold switches is their background signal (translation in absence of trigger sequence), so we tested only one trigger concentration.

Initially we tested Toehold 14, which was less promising than Toehold 13 according to the *in silico* analysis. Our results confirmed the *in silico* prediction, as Toehold 14 was unable to control translation, compared to controls (Fig. 19).

![](https://static.igem.org/mediawiki/2019/d/db/T--Thessaly--Results_Figure22.jpg)

**Figure 19.** Bars showing the performance of toehold 14 to hydrolyze nitrocefin.

As seen in Figure 19, the signal produced by Toehold 14 is very similar to that of the positive control. However, when no trigger sequence is added, the signal produced is the same as the 100nM trigger condition. This can be interpreted as the toehold 14 ‘s inability to control translation. That means that the toehold switch has an open conformation and thus an accessible RBS, even when no trigger is added.

Since testing toehold 14 resulted in a failure, we turned our interest to Toehold 13. Following the same conditions as before, we assessed the performance of toehold 13 in the same β-lactamase enzymatic assay. The results this time were more encouraging, as depicted in Figure 20.

![](https://static.igem.org/mediawiki/2019/3/33/T--Thessaly--Results_Figure23.jpg)

**Figure 20.** Performance of toehold 13 in a β-lactamase enzymatic assay. Error bars represent standard deviation of n = 2 replicates. Blank was subtracted.

The expression of β-lactamase under the regulation of Toehold 13 was noticeably low in the absence of the trigger sequence, while the 100nM trigger reaction easily reached levels of signal comparable to the positive control. However, when compared to the literature 32B toehold[4], the background signal of toehold 13 was ~3-fold higher. This is sensible, since a more thorough *in silico* and *in vitro* screening was conducted in the paper, resulting in around 10 times more toehold switches that were tested.

Even if not perfectly functional, toehold 13 looks promising as a universal toehold switch. Furthermore, it is amenable to optimization both *in silico* and *in vitro*, something that we did not manage to do due to time limitations. We aspire that we or future teams will take the foundation we have provided and optimize this promising part into something high performing.

**Future vision**

Besides the above mentioned results and the goals we have already achieved, there are some aspects of our project we could explore more in the future, to improve it and eventually make it as efficient as possible.

**SAMRS**

Some future plans for us to test would be the redesign of our primer sets using SAMRS nucleotides, so that the RPA reaction would get no non-specific products amplified along with the specific one. Following reported literature on RPA-SAMRS primers, we have already [**re-designed**](https://2019.igem.org/Team%3AThessaly/Model) our primer sets accordingly. This way, we hope to reduce the primer-dimer formation that we encountered in our non-SAMRS primers. This plan was initially part of our experimental design, however, the cost for the purchase of these primer sets was too high for an iGEM team.

**Redesign our universal toehold switches**

As we mentioned above, we have [**designed**](https://2019.igem.org/Team%3AThessaly/Model)a pool of universal toehold switches from hyperthermophile bacteria *in silico*, but due to budget limitations, only the two most promising switches were ordered and tested, according to the *in silico* analysis. An easily achievable future plan for us, would be the screening of a larger pool of toehold switches (similar to the design principles of the ones we tested) and the assessment of their efficiency until we find the one that can provide us with the unique element of universality as well as with the highest possible efficiency.

**Test the positive control**

The nature of our design is such, that a positive control is needed for the correct functionality of our diagnostic test. This is why we chose the cox3 gene deriving from the human mitochondrial DNA, for a [**positive control**](https://2019.igem.org/Team%3AThessaly/Design), which is also included in our product design and will be incorporated into our final product. A future plan would be to actually redesign our primers to be able to target this gene and test if we can detect it and amplify it.

**Synthetic urine**

During the iGEM competition, we discovered that safety is of utmost importance. Due to this fact we did not use any urine samples to test the efficiency of our design. A future plan for us would be to spike synthetic urine with the fragmented biomarker we chose to target (*IS6110* or HBV) and check if we would be able to detect it after a pre-treatment step of the sample. This way, we will be able to simulate, as much as possible, realistic conditions for our diagnostic test.

**Cell lysate**

In order to minimize the cost of our diagnostic test, we aim to create cell-lysates for the *in vitro* transcription/translation system. Cell-free expression systems can be prepared using either a cell extract, or a combination of purified recombinant proteins, and be done at 37 – 42℃ optimally. As it would be a very important upgrade of our design, it would be crucial for us in the future, to prepare our own cell-free expression system (cell lysate) and test its efficiency.

**References**

1.Labugger I, Heyckendorf J, Dees S, Häussinger E, Herzmann C, Kohl T A, Richter E, Milla E R, Lange C. Detection of transrenal DNA for the diagnosis of pulmonary tuberculosis and treatment monitoring, *Infection* (2016). doi:10.1007/s15010-016-0955-2

2. I.M. Lobato, C.K. O’Sullivan, Recombinase Polymerase Amplification: Basics, applications and recent advances, *Trends in Analytical Chemistry* (2017)

3.Sharma, N., Hoshika, S., Hutter, D., Bradley, K. M., & Benner, S. A. (2014). Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS). *ChemBioChem*, 15(15),2268–2274. doi: 10.1002/cbic.201402250

4.
Pardee Keith, Alexander A Green, Melissa K Takahashi, David H O Connor, Lee Gehrke, James J Collins (2016). “Rapid , Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components ” *Cell*, 1–12. https://doi.org/10.1016/j.cell.2016.04.059.

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