iGEM 2018 — Team Athens

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Athens/Notebook

* [![](https://static.igem.org/mediawiki/2018/0/01/T--Athens--iGEMAthensLogo.png)](https://2018.igem.org/Team%3AAthens)
* Team

  [Members](https://2018.igem.org/Team%3AAthens/Team)
  [Collaborations](https://2018.igem.org/Team%3AAthens/Collaborations)
  [Attributions](https://2018.igem.org/Team%3AAthens/Attributions)
* Project

  [Description](https://2018.igem.org/Team%3AAthens/Description)
  [MERS-CoV](https://2018.igem.org/Team%3AAthens/MersCov)
  [Notebook](https://2018.igem.org/Team%3AAthens/Notebook)
  [Design](https://2018.igem.org/Team%3AAthens/Design)
  [Results](https://2018.igem.org/Team%3AAthens/Results)
  [Safety](https://2018.igem.org/Team%3AAthens/Safety)
* Wet Lab

  [Experiments](https://2018.igem.org/Team%3AAthens/Experiments)
  [InterLab](https://2018.igem.org/Team%3AAthens/InterLab)
  [Parts](https://2018.igem.org/Team%3AAthens/Parts)
* Dry Lab

  [Toehold Design](https://2018.igem.org/Team%3AAthens/Model)
  [Kinetic Modeling](https://2018.igem.org/Team%3AAthens/Model#kinetic)
  [3D Simulations](https://2018.igem.org/Team%3AAthens/Model#3d_simulations)
  [Temperature Simulations](https://2018.igem.org/Team%3AAthens/Model#temp_simulations)
* Human Practices

  [Overview](https://2018.igem.org/Team%3AAthens/HP_Overview)
  [Education & Public Engagement](https://2018.igem.org/Team%3AAthens/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AAthens/Human_Practices)
* Product

  [Applied Design](https://2018.igem.org/Team%3AAthens/Applied_Design)

Interlab Study

# Notebook

## May

Getting familiarized with laboratory techniques and planning a timeline. Maria and Natalia will be primarily working at the Molecular Virology Laboratory of the Hellenic Pasteur Institute. Nelly, Elena and Panos will be mainly working at the Biotechnology Laboratory of NTUA and will be in charge of the Interlab study.

## June

### Hellenic Pasteur Institute

* Competent cells protocol (DH5a, BL21)
* Transformation and first trial with the Monarch® Plasmid Miniprep Kit | NEB using pGEM-luc as test plasmid
* Plasmid digestion using Xmnl, Xbal
* Getting familiar with agarose gel electrophoresis
* Gel extraction using Monarch® DNA Gel Extraction Kit | NEB
* Trehalase (TreA) isolation from BL21 E.coli cells
* DNA isolation using the QIAGEN® Plasmid Maxi Kit.

### NTUA

* Interlab: followed the iGEM competent cells protocol, performed the competent cell test, with the kit provided by iGEM

## July

### Hellenic Pasteur Institute

* Design of primers for TreA, TreA-a, and TreA-b and selection of enzymes that will be used
* Design of leucine zippers for protein- protein interaction
* Digest of pET-15b and pGEM3zf(+) with NdeI, BamHI and EcoRI respectively
* Agarose gel electrophoresis for the pET-15b digest, gel extraction, Maxiprep of pET-15b using the QIAGEN® Plasmid Maxi Kit and transformation.

### NTUA

* Unsuccessfully tried to transform competent cells with a GFP part found in the kit, aiming to use GFP as an alternative reporter protein for our system.
* While troubleshooting we tested the competence of the cells and concluded the error lied in the GFP plasmid.
* Interlab: Transformation with the plasmids provided by the iGEM Distribution Kit. OD and fluorescence measurements of our overnight cultures. Measurement of the fluorescence with Flow Cytometry. Followed the CFU protocol and counted colonies.

## August

### Hellenic Pasteur Institute

* PCRs using the Q5® High-Fidelity DNA Polymerase (M0491) | NEB and the Phusion® High-Fidelity DNA Polymerase | NEB
* PCR for TreA, phenol extraction of the pET-15b maxiprep DNA, double digestion of the pET-15b vector and the pure whole TreA using NdeI, BamHI and ligation of the pET-15b vector and TreA
* Transformation of pET-15b & TreA ligation product and minipreps. 1:10 ligation worked the best. Maxiprep of “plasmid A” (plasmid A= pET-15b vector + TreA insert). Acquisition of TreA expression plasmid.
* Purification of trehalase using the Protino® Ni-TED and Ni-IDA Packed Columns, in both native & denaturing conditions. Best results observed using the Ni-IDA column, in native conditions.
* PCRs for the TreA-a and TreA-b parts

## September

### Hellenic Pasteur Institute

* Elena joins the Pasteur lab team
* Polyacrylamide gel electrophoresis (10% (w/v) SDS PAGE) under reducing conditions, staining with coomassie blue.
* Benedict’s tests trials were performed using glucose solutions.
* Benedict’s, DNS tests were performed. DNS was proved to have greater sensitivity.
* Linearization of the maxi prepped pGEM vector via PCR, assembly of pGEM vector, Toehold Switches 3,7,8,16 and TreA-a,. Transformation. No colonies.
* Linearization PCR for triggers 3,7,8,16 vectors, PCR purification and assembly of triggers 3,7,8,16. Transformation of the digested products. No colonies.
* Assembly of zipper-b and TreA-b and transformation. No colonies.
* Tried gel extraction of the assembled product for all the unsuccessful transformations. No colonies.
* PCR cleanup kit on TreA whole, PCR linearization and deletion for Toehold switch 16 (TS plasmid + treA-a), confirmation PCR, assembly of TS and TreA. Transformation. No colonies.
* Again tried transformation after gel extraction of the assembled product and ethanol precipitation in both DH5a and XL1-Blue strains. Still no colonies.
* Tried transforming the gel extracted assembled vector after ethanol precipitation for every assembly product. Successful transformation in both DH5a and XL1-Blue strains (even though low number of colonies), for Toehold Switches 7,8,16, triggers 3, 16, zipper-b/ TreA-b. Colonies did not contain correct parts, except in the case of Toehold Switch 16.
* Plasmid DNA extraction and confirmatory digest (with PvuII for toehold switches, PvuII and EcorI for trigger RNAs, XbaI and SpeI for zipper-b/ TreA-b). Turns out that only Toehold Switch 16 and zipper-b/ TreA-b were successful.

## October

### Hellenic Pasteur Institute

* Nelly joins the Pasteur lab team.
* Digests of Toehold Switch 16 + TreA-a and igem vector using XbaI, SpeI. Dephosphorylation on igem vector using CIAP, to prevent self-ligation. Ligation on three different ratios. Transformation in both DH5a and XL1-Blue strains. No colonies.
* Transformation of the ligated product after gel extraction and ethanol precipitation. No colonies.
* Started the ligation process again. Still no colonies.
* At last, we obtained colonies from the ligation of part but didn’t have enough time to screen.
* Repeated successfully Trigger 16 assembly and transformation with freshly-made competent cells but there was not enough time to screen.
* Expression of TreA with the PURExpress® In Vitro Protein Synthesis Kit | NEB, in order to recreate the testing part. We used DNS and a glucometer to find out the time required for diagnosis.

### Contact Info:

[![Link to our Facebook page](https://static.igem.org/mediawiki/2018/3/30/T--Athens--FB-logo.png)](https://www.facebook.com/iGEM.Athens.18/)
[![Link to our Instagram page](https://static.igem.org/mediawiki/2018/c/c3/T--Athens--Instagram-logo.png)](https://www.instagram.com/igemathens2018/)
[![Link to our Twitter page](https://static.igem.org/mediawiki/2018/a/a2/T--UCL--twitter1.png)](https://twitter.com/igemathens2018)

igemathens2018@gmail.com


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Athens/Experiments

* [![](https://static.igem.org/mediawiki/2018/0/01/T--Athens--iGEMAthensLogo.png)](https://2018.igem.org/Team%3AAthens)
* Team

  [Members](https://2018.igem.org/Team%3AAthens/Team)
  [Collaborations](https://2018.igem.org/Team%3AAthens/Collaborations)
  [Attributions](https://2018.igem.org/Team%3AAthens/Attributions)
* Project

  [Description](https://2018.igem.org/Team%3AAthens/Description)
  [MERS-CoV](https://2018.igem.org/Team%3AAthens/MersCov)
  [Notebook](https://2018.igem.org/Team%3AAthens/Notebook)
  [Design](https://2018.igem.org/Team%3AAthens/Design)
  [Results](https://2018.igem.org/Team%3AAthens/Results)
  [Safety](https://2018.igem.org/Team%3AAthens/Safety)
* Wet Lab

  [Experiments](https://2018.igem.org/Team%3AAthens/Experiments)
  [InterLab](https://2018.igem.org/Team%3AAthens/InterLab)
  [Parts](https://2018.igem.org/Team%3AAthens/Parts)
* Dry Lab

  [Toehold Design](https://2018.igem.org/Team%3AAthens/Model)
  [Kinetic Modeling](https://2018.igem.org/Team%3AAthens/Model#kinetic)
  [3D Simulations](https://2018.igem.org/Team%3AAthens/Model#3d_simulations)
  [Temperature Simulations](https://2018.igem.org/Team%3AAthens/Model#temp_simulations)
* Human Practices

  [Overview](https://2018.igem.org/Team%3AAthens/HP_Overview)
  [Education & Public Engagement](https://2018.igem.org/Team%3AAthens/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AAthens/Human_Practices)
* Product

  [Applied Design](https://2018.igem.org/Team%3AAthens/Applied_Design)

Experiments

* [• Trehalase](#trehalase)
* [• Gibson Assembly](#gibson)
* [• Toehold Switch Testing](#testing)
* [• Protocols](#protocols)

# Experiments

## Trehalase

![](https://static.igem.org/mediawiki/2018/4/4f/T--Athens--exp-flow1.png)

The first step of our experimental process was the isolation of our reporter gene, trehalase (TreA) from E.coli BL21 genomic DNA. We chose to clone TreA in pET-15b vector, since pET systems are reliable for recombinant protein expression in E. coli. After cloning TreA successfully, we used the whole gene to PCR amplify the two split fragments (TreA-a & TreA-b). The expression and subsequent purification of TreA was a prerequisite in order to gain insight on the trehalase assay and optimize its conditions. The 6xHIS-tagged protein was purified with nickel columns, in native and denaturing conditions. The denaturing agent used was guanidine.

![](https://static.igem.org/mediawiki/2018/f/f2/T--Athens--exp-flow2.png)

The evaluation of the enzymatic activity of the purified protein followed, using a glucose detection assay. Trehalase hydrolyses the disaccharide trehalose to glucose, which, in our case was detected by two different methods DNS and Benedict’s test, in order to assess which one was more sensitive and ensure a result in variable conditions. In the first instance, we assayed trehalase activity at pH 7.5, which resulted in no enzymatic activity. That is because the catalytic activity of trehalase is optimized at a slightly acidic pH. Thus, we continued our experiments fixing the trehalase assay pH at 5.9. We incubated purified trehalase with trehalose for 15min at 37oC and then added either DNS or Benedict’s reagent to the reaction.

Both methods are based on color change. The DNS method produces a change from light to dark orange, while the Benedict’s test changes from clear blue to cloudy red (blue-green-yellow-orange-red). As is evident from the above image, the DNS method gave better results, with obvious darkening in the color of all the protein samples’ reactions. Concluding, we decided to perform our toehold switch testings with the trehalase assay at pH 5.7 and using DNS for glucose detection.

## Gibson Assembly

The cloning vector chosen for our trigger RNAs and for our toehold switches was pGEM-3zf+. The main reason for this decision was the fact that this vector contains a T7 promoter, which would facilitate the in-vitro transcription of our parts. After each vector linearization, we performed treatment with the restriction enzyme DpnI, in order to destroy the plasmid template before setting up the NEBuilder HiFi DNA assembly reaction.

As mentioned above, the sequences of TreA used were isolated from the genome of *E. coli* BL21. The toehold switches that contain the corresponding zipper sequence, along with the triggers were all synthesized as double-stranded DNA fragments (IDT) .

### Trigger RNAs

![](https://static.igem.org/mediawiki/2018/9/93/T--Athens--exp-flow3.png)
![](https://static.igem.org/mediawiki/2018/3/34/T--Athens--exp-trigger.png)

Our trigger primers were 30 nt long, with vector overlaps of 25-30 nt on both ends. Thus, preparation for the assembly only demanded linearization of the vector.

### Toehold Switches with TreA-a

![](https://static.igem.org/mediawiki/2018/f/f5/T--Athens--exp-flow4.png)
![](https://static.igem.org/mediawiki/2018/9/9f/T--Athens--exp-treaA.png)

In this case, there were three fragments to be assembled: the vector, the toehold switch (from TBS up to zipper-a) and TreA-a. Therefore, we needed to perform two PCR reactions as preparation for the assembly; one for the linearization of the vector and one for the attachment (ligation) of overlapping primers at the ends of TreA-a. Having overlaps on the ends of all the fragments to be assembled, we could perform the cloning.

### Toehold Switches with TreA

![](https://static.igem.org/mediawiki/2018/6/61/T--Athens--exp-flow5.png)
![](https://static.igem.org/mediawiki/2018/4/46/T--Athens--exp-trea.png)

For the toehold switches with whole trehalase as reporter (TreA), we used as template vectors the assembled vectors with TreA-a which were produced in the previous step for each toehold switch. At the linearization step, the primers were designed so that the amplified part would be the whole vector apart from the zipper-a and TreA-a sequence. This way, this part would be deleted and we could replace it with TreA in the assembly process, constructing the corresponding toehold switches with TreA as reporter.

### Zipper-b and TreA-b

![](https://static.igem.org/mediawiki/2018/7/7d/T--Athens--exp-flow6.png)
![](https://static.igem.org/mediawiki/2018/1/1e/T--Athens--exp-treaB.png)

For this assembly, the fragments we worked with were zipper-b, TreA-b and pET-15b vector. We followed the same process steps as in the assembly of the switches. The only alteration was the design of a ds-DNA region on the 5’ end of zipper-b overlapping the vector, which was used instead of overlapping primers.

### Cloning Evaluation

![](https://static.igem.org/mediawiki/2018/9/9c/T--Athens--exp-flow7.png)

After each assembly, E. coli DH5a chemically competent cells were transformed with the produced parts. Initially, following the protocol of NEBuilder HiFi DNA Assembly Cloning Kit, we used the assembly product mixture for the transformation, which resulted in no colonies. The first alternative we tried was gel extracting the product (via Monarch® DNA Gel Extraction Kit, NEB), in order to transform the cells solely with the assembled vector. This too resulted in no colonies. We also tried performing DNA purification to the assembly product mixture (via Monarch® PCR & DNA Cleanup Kit, NEB), getting the same result. Attempting to solve this problem, we performed the same transformations using XL1-Blue competent cells as well, which again resulted in no colonies. In the above cases we could detect the correct products following electrophoresis of the assembly reaction.

![](https://static.igem.org/mediawiki/2018/8/84/T--Athens--exp-trivlio.png)

Therefore, we decided to concentrate the ligated DNA by ethanol precipitation before transformation. This method was successful for some assembly reactions in both DH5a and XL1-Blue strains, although the growth rate of the forming colonies was relatively small.

After screening these colonies by extracting plasmid DNA with small scale preparation (mini preps) and the appropriate enzymatic digests, we obtained the assembled sequences for Toehold Switch 16 and zipper-b/ TreA-b. We were unable to obtain the assembled sequences of the toehold switches with the whole trehalase, the triggers, and other toeholds with the split trehalase. One possible reason for not being able to obtain the switch with the whole trehalase is the fact that the insert was relatively large, resulting in low yield in the assembly process. In our effort to solve this issue, we increased the assembly time (4 hours instead of 50 minutes), which nevertheless did not work. Regarding the triggers, we tested the very few colonies we obtained but they contained only the empty vector. These transformations should be repeated in order to get more colonies for screening.

## Toehold Switch Testing

![](https://static.igem.org/mediawiki/2018/e/e5/T--Athens--exp-flow8.png)

Recapping, for each toehold switch testing, the corresponding trigger RNA represents a sequence of the viral RNA, which would be amplified during the sample pretreatment. In the in-vitro transcription & translation system, the toehold switches are transcribed by default, but their translation is only allowed upon trigger binding, under the condition that the toehold switch - trigger RNA pair tested is successful.

Thus, adding the transcribed trigger RNA in the transcription & translation system, was expected to activate TreA-a / zipper-a expression. Sampling from this reaction and mixing this sample with TreA-b / zipper-b, would allow zipper interactions and consequently TreA reassembly. The TreA-b / zipper-b complex would be expressed using the same kit, in order to avoid any buffer incompatibilities.

### Trehalase Cell-Free expression

Since we did not have enough time to solve our transformation problem and did not obtain all the parts needed for the toehold switch testing, we decided to get a rough estimate of the time needed for a diagnosis by expressing TreA in our cell-free system. Therefore, we set up a PURExpress® reaction using 1 μg of TreA expression plasmid and carried out coupled transcription / translation for 3.5 hours to ensure sufficient TreA protein production. We tested the resulting reaction for the presence of functional TreA by incubating a small quantity of the transcription/translation reaction with trehalose and then adding DNS reagent. As shown in the photo below, appropriate colour formation indicated that the pH of the reaction was suitable for the TreA to work.

![](https://static.igem.org/mediawiki/2018/f/f9/T--Athens--exp-katoura1.png)

**Left**: Blank without trehalose. **Right**: Blank with trehalose

We then tried to determine the minimum time demanded for sufficient TreA production in order to reach the DNS glucose threshold. Thus, we took samples from the reaction at different time points and performed the same process as before.

![](https://static.igem.org/mediawiki/2018/a/a3/T--Athens--exp-katoura2.png)

We concluded that about 1 hour of **TreA expression** was demanded to produce sufficient TreA, in order to get a visible color change in the DNS assay.

### Commercial Glucose Meter Results

The commercial glucose meter has a lower sensitivity, since it is intended for the quantification of glucose in human blood samples, on which the levels are significantly higher compared to the threshold of the DNS assay. Thus, performing the same experiments as for the DNS assay, we first attempted to prove that the time frame of 3.5 hours of reaction could give sufficient amount of TreA, in order to be subsequently quantified by the glucose meter.

![](https://static.igem.org/mediawiki/2018/f/f7/T--Athens--exp-meter1.png)

The results confirmed that the commercial glucose meter is a suitable signal indicator and most importantly, that our proposed diagnostic methodology is valid. On the same logic as in the DNS assay experiments, we defined the minimum translation time needed in order to get enough TreA and a detectable signal from the commercial glucose test.

![](https://static.igem.org/mediawiki/2018/d/d3/T--Athens--exp-meter2.png)

The samples taken at t=0, 45min, 1hr, 1:15hr, 1:30hr resulted in this indication, which implies that the glucose concentration was too low to be quantified.

![](https://static.igem.org/mediawiki/2018/6/6d/T--Athens--exp-meter3.png)

The first measurement received was at the time frame of 1:45 hours post **TreA expression**.

❮
❯

### iGEM Part Construction

We chose XbaI and SpeI restriction enzymes that were included in the prefix and suffix of the pSB1C3 vector. We incorporated these enzymes in our overlapping primers so that all parts could be cloned directly into the pSB1C3 vector. We digested parts and vector with the enzymes and then we dephosphorylated pSB1C3 with Calf Intestine Alkaline Phosphatase (CIAP) to prevent vector religation since XbaI and SpeI create compatible overhangs. We carried out several ligations with T4 ligase with various ratios of vector to insert. After several unsuccessful transformations, we obtained red colonies but one day after the part submission deadline. Therefore, we were unable to send our part in the pSB1C3 vector.

## Protocols

### All Protocols were carried out following the manufacturer’s instructions.

* **Minipreps** were carried out using the Monarch® Plasmid Miniprep Kit | NEB.
* **Maxipreps**were carried out using the QIAGEN® Plasmid Maxi Kit.
* **Gel extractions**were carried out using the Monarch® DNA Gel Extraction Kit | NEB.
* **PCR purifications**were carried out using the Monarch® PCR & DNA Cleanup Kit (5 μg) | Purification Kits | NEB.
* **Assemblies** were carried out using the NEBuilder® HiFi DNA Assembly Cloning Kit | NEB.
* The Protino® Ni-TED and Ni-IDA Packed Columns - MACHEREY-NAGEL were used for the **purification of His-tag proteins.**
* The PURExpress® In Vitro Protein Synthesis Kit | NEB was used for **in vitro transcription| translation.**

### Preparation of competent cells

1. Pick a single colony from a fresh LB-plate. Grow overnight at 37oC (10 ml in a universal).
2. Transfer 5 ml of overnight cells into 100 ml of LB in a 500 ml flask. Grow with shaking at 37oC for 2-3 hours, until the OD600 reaches 0.4- 0.6.
3. Chill the cells on ice for 5 min. Transfer to chilled Falcon tubes and spin at 4oC at about 3K for 5 mins.
4. Pour away the supernatant. Resuspend gently in 25 ml of cold 0.1 M CaCl2. Leave it on ice for 2 min.
5. Gently spin for 10 mins at 3000 rpm at 4oC.
6. Resuspend in cold 0.1 M CaCl2 (10 ml) and leave for at least 1 hour on ice (1-4 hours).
7. Add 10 ml of 40% sterile glycerol and mix gently.
8. Transfer 1 ml into chilled Eppendorf tubes.
9. Freeze at -80oC.

### Transformation

1. Prepare LB+ antibiotic agar plates.
2. Take out the competent cells from the freezer and place on ice (30 min- 1 hour).
3. Aliquot into 200 μl aliquots in Eppendorf tubes.
4. Add 1-5 μl of ligation/ DNA to each tube. Mix gently. Leave on ice for 40 min.
5. Heat shock cells for 90 sec at 42oC. Place on ice for 2 min.
6. Add 800 μl of LB medium prewarmed to 37oC.
7. Grow gently shaking for 1 hour (37oC).
8. After incubation for 1 hour at 37oC, 100 μl of culture can be spread on the plate and the rest of the transformation can be centrifuged for 3 min at 13000 rpm. Subsequently, supernatant is discarded and the pellet is resuspended in 100 μl of LB. This solution is spread on the plate.
9. The plates are left to dry for 10- 15 min. After drying they are incubated at 37oC in inverted position.

### LB Agar preparation

1. Measure 500 ml of ddH2O and transfer in glass bottle.
2. Weigh 15.25g of Luria Agar powder and add to water.
3. Autoclave the bottle containing the solution.
4. When the solution has cooled down, add antibiotic.
5. Pour the LB Agar solution into plates.

### LB Broth preparation

1. Measure 500 ml of ddH2O and transfer in glass bottle.
2. Weigh 5 g tryptone, 2.5 g yeast, 2.5g NaCl and add to water.
3. Autoclave the bottle containing the solution.

### Glycerol stock preparation

1. Pour 500 ul of glycerol 40% in 1.5 or 2 ml tube.
2. Add 500 ul of culture in LB.
3. Store at -80oC.

### Overnight inoculums

1. Add 5 ml of LB broth to glass tubes.
2. Add antibiotic (volume depends on antibiotic’s concentration in order to reach the correct final concentration).
3. Pick single colonies from plate using a pipette with a tip on and inoculate the LB.

### Agarose gel

Agarose gel % and volume were chosen according to the needs of each electrophoresis performed. For an 1% agarose gel (100 ml):

1. Measure 100 ml of 1X TBE and transfer to a Pyrex glass bottle.
2. Weigh 1g of agarose and add to bottle.
3. Heat mixture in the microwave until agarose is totally dissolved.
4. Carefully add 7 ul of EtBr.
5. Pour mixture into cast.

### Agarose gel electrophoresis

1. Place agarose gel previously prepared in agarose gel electrophoresis device.
2. Fill the apparatus with TBE 1X.
3. Load appropriate ladder and the samples to the wells of the gel.
4. Place the lid on the device, connect the electrodes and run the gel.
5. Turn off power and disconnect electrodes.
6. Assess bands under UV light.

### Polyacrylamide gel electrophoresis

For a 10% SDS PAGE:

* For a 5 ml stacking gel: 3.4 ml H2O, 1.25 ml 0.5 M Tris-HCl (pH 6.8), 0.05 ml 10% (w/v) SDS, 0.83 ml 30% Acrylamide, 50 ul 10% (w/v) AP, 5 ul TEMED
* For a 10 ml separating gel: 4 ml H2O, 3.3 ml 30% Acrylamide, 0.63 ml 1M Tris (pH= 8.8), 0.05 ml 10% (w/v) SDS, 0.05 ml 10% (w/v) AP, 5 ul TEMED

1. Make the separating gel.
2. Set the cast.
3. Carefully pipette the gel in the gap between the glass plates of the cast.
4. Make the stacking gel.
5. When separating gel is ready, carefully pipette in the stacking gel on top.
6. Place comb.
7. When gel is ready, take out comb, set the glass frames in the running device and pour in the running buffer.
8. Prepare and load samples under reducing conditions.
9. Connect the electrodes and run the electrophoresis.
10. Turn of power and disconnect the electrodes.
11. Assess bands by staining with Coomassie blue and de-staining the gel.

### Enzymatic reactions

**Digests** were carried out at 50 ul using 5 units of enzyme, x μl of DNA (depending on each digest), 1x digest buffer, x ul WFI (depending on each digest). Digests were placed in the thermocycler at 37oC for 3 hours or overnight, depending on each digest. Heat inactivation of the enzymes was performed for 20 minutes at 65 or 80oC, depending on each digest.

**Ligations** were performed following the T4 DNA Ligase NEB protocol, at different ratios of vector/insert, always following the manufacturer’s instructions.

### Contact Info:

[![Link to our Facebook page](https://static.igem.org/mediawiki/2018/3/30/T--Athens--FB-logo.png)](https://www.facebook.com/iGEM.Athens.18/)
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igemathens2018@gmail.com

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# Team:Athens/Results

* [![](http://2018.igem.org/wiki/images/0/01/T--Athens--iGEMAthensLogo.png)](http://2018.igem.org/Team%3AAthens)
* Team

  [Members](http://2018.igem.org/Team%3AAthens/Team)
  [Collaborations](http://2018.igem.org/Team%3AAthens/Collaborations)
  [Attributions](http://2018.igem.org/Team%3AAthens/Attributions)
* Project

  [Description](http://2018.igem.org/Team%3AAthens/Description)
  [MERS-CoV](http://2018.igem.org/Team%3AAthens/MersCov)
  [Notebook](http://2018.igem.org/Team%3AAthens/Notebook)
  [Design](http://2018.igem.org/Team%3AAthens/Design)
  [Results](http://2018.igem.org/Team%3AAthens/Results)
  [Safety](http://2018.igem.org/Team%3AAthens/Safety)
* Wet Lab

  [Experiments](http://2018.igem.org/Team%3AAthens/Experiments)
  [InterLab](http://2018.igem.org/Team%3AAthens/InterLab)
  [Parts](http://2018.igem.org/Team%3AAthens/Parts)
* Dry Lab

  [Toehold Design](http://2018.igem.org/Team%3AAthens/Model)
  [Kinetic Modeling](http://2018.igem.org/Team%3AAthens/Model#kinetic)
  [3D Simulations](http://2018.igem.org/Team%3AAthens/Model#3d_simulations)
  [Temperature Simulations](http://2018.igem.org/Team%3AAthens/Model#temp_simulations)
* Human Practices

  [Overview](http://2018.igem.org/Team%3AAthens/HP_Overview)
  [Education & Public Engagement](http://2018.igem.org/Team%3AAthens/Public_Engagement)
  [Integrated Human Practices](http://2018.igem.org/Team%3AAthens/Human_Practices)
* Product

  [Applied Design](http://2018.igem.org/Team%3AAthens/Applied_Design)

GENOMERS Results

# Results

### Project: MERS-CoV molecular diagnosis via the toehold switch mechanism and glucose monitoring

## Achievements

### Detection Mechanism: Toehold Switch

![](http://2018.igem.org/wiki/images/9/91/T--Athens--ToeholdStructure.jpeg)

1. Developed a computational tool for predicting mRNA sequences with optimal toehold switch functionality.
2. Successfully constructed a SynBio part in a pGEM vector, containing a toehold switch regulated by a specific and conserved MERS-CoV genomic region and encoding split trehalase - part A.

### Reporter System: Trehalase & Split Trehalase

![](http://2018.igem.org/wiki/images/f/f3/T--Athens--project-design-trea%2Btreb.gif)

1. Proposed an innovative reporter system for semi-quantitative diagnosis, incorporating the enzyme trehalase as well as a split trehalase system expected to interact via coiled coil interactions.
2. Successfully predicted the chimeric protein structures of our split trehalase system (part A and part B), via Homology Modeling.
3. In order to gain insight on the split trehalase system behaviour, we performed Molecular Dynamics Simulations, with promising results.
4. Studied the kinetics of the cell-free system and developed a consistent kinetic model for the trehalase coupled transcription/translation and the enzymatic reaction.
5. Tested the TreA enzymatic assay with the DNS method and with a common glucometer, both showing promising results for rapid POC diagnosis.

### Applied Design: Diagnostic Kit

1. Proposed the detailed design of a diagnostic kit for POC diagnosis, with significant competitive advantages to existing diagnostic methods (qRT-PCR).
2. Developed a 3D model of the diagnostic kit for the study of the temperature profile, calculating proper times and dimensions for optimum performance.

### Overview: Our overall achievements

![](http://2018.igem.org/wiki/images/e/e9/T--Athens--project-design-project-linear.gif)

1. Monitored the cell-free trehalase-produced glucose with a commercial glucometer.
2. Concluded that detectable trehalase amounts are produced in the time frame indicated by the kinetic model, indicating that trehalase could be a suitable reporter protein for molecular diagnostics.
3. Demonstrated that the proposed diagnostic method can be performed in under one hour, therefore being suitable for Point-of-Care diagnosis.
4. Successfully completed the InterLab Study

### Human Practices: Engaging with the world

1. Performed a series of educational and public-engagement-oriented activities, entitled “Synthetic Biology: The Principles of Engineering Life.”
2. Gathered our observations on the presence of Synthetic Biology in the Greek reality in an elaborate report. Further discussed our findings with specialists and academics, and documented our suggestions.
3. Created a video addressing high school students that took a linguistic approach on explaining Synthetic Biology.
4. Documented the procedure of organizing a SynBio Workshop on a guide for future iGEM reference.
5. Got in contact with specialists from all over the world and gained valuable feedback that we incorporated in our project, both in wet lab and dry lab experiments, as well as in our suggested diagnostic method and final product design.
6. Collaborated with various iGEM-ers from around the world and engaged in meaningful discussions with the scientific community of Synthetic and Systems Biology.

## Complications

1. We encountered difficulties in transforming the trigger parts, therefore we could not assess the functionality of the constructed toehold.
2. We only produced a toehold switch with the split trehalase as a reporter protein.
3. The constructed toehold switch could not be inserted in the pSB1C3 vector. The ligation product was finally transformed and minipreped, however, after digestion, no bands were detectable on the gel.
4. The Toehold Switch Molecular Dynamics simulation was an aspiration that remained fruitless, as RNA modelling is a demanding field where limited softwares are available for producing promising results.

## Future Goals

1. Design and perform a trehalase quantification assay and establish a viral load- protein production correlation.
2. Create and test toehold switches containing the whole trehalase and evaluate this reporter protein relatively to beta-galactosidase.
3. Apply the toehold switch detection mechanism coupled with the glucose monitoring test for a series of other viruses such as West Nile virus and coinfection cases such as Hepatitis C and B coinfection.
4. Evaluate the potential parallel use of different toehold-switches targeting different viral genomic regions for more reliable diagnosis.
5. Create a 3D model of the 2D studied toehold switches.
6. Investigate other methods for the leucine zippers in silico study and the assembled trehalase molecule modeling.
7. Validate the current model with experimental data and develop a new kinetic model for the split trehalase reporter.
8. Build and assess a prototype of the proposed diagnostic kit.

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