iGEM 2022 — Team tau-israel

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**Toeholds testing – Mammalian cell-lines**
**Toeholds Testing – Yeast**
**Parts’ Optimization**

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## Toeholds Testing – Mammalian Cell-lines

---

* Lab space and instrumentation for working with mammalian cell lines were provided courtesy of Prof. Dan Peer’s laboratory ([Lab website](https://dan-peer.tau.ac.il/)).* Routine handling of the mammalian cell lines is not specified in the lab notebook (cells were grown in complete DMEM and handled according to standard procedures (see [ATCC guidelines](https://www.atcc.org/products/crl-1573)).
  * Our Protocols can be found [here](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Lab_protocols.pdf)

**May 1, 2022**

* [miRNA-155](https://www.addgene.org/78126/) and [miRNA-21](https://www.addgene.org/21114/) expression plasmids ordered.
* Chemically induced competent E. coli (D5H) were transformed via heat-shock with Wang and colleagues' miR-155 Toehold plasmid1, provided by courtesy of Shue Wang’s Laboratory at UNH, ([Lab website](https://www.newhaven.edu/faculty-staff-profiles/shue-wang.php)) and with the two miR plasmids and were then plated in LB-ampicillin plates.

**May 2, 2022**

* Colonies from plates were collected into LB+Amp.

**May 3, 2022**

* Plasmids were extracted from tubes and sent for Sanger sequencing (using the T7 primer).
* 25% glycerol stocks were made and put in -80oc.

**May 9, 2022**

* HEK293 cells were seeded in a 24-well plate (2\*105 cells/well), to prepare for an evaluation of Wang and colleagues’ toehold (1).

**May 10, 2022**

* Cells were transfected with either miR-155 toehold alone or with miRNA-155 expression plasmid and the toehold together.
* 100ng DNA from each plasmid was used for this experiment.

**May 11, 2022**

* Cells were collected, washed with PBS, and run through FACS analysis via CytoFLEX Flow Cytometer (Beckman Coulter, CA, USA).
* Exhibited no GFP expression in any condition, therefore higher DNA concentrations were used for latter experiments.

**May 15, 2022**

* HEK293 cells were seeded in a 24-well plate for another test of Wang and colleagues’ toehold (1).

**May 16, 2022**

* Cells were transfected with either miR-155 toehold or with miRNA-155 expression plasmid and the toehold together (400 ng DNA from each plasmid).

**May 17, 2022**

* Cells were collected, washed with PBS, and run through FACS analysis.
* Exhibited no GFP expression in any condition, therefore DNA concentrations were further elevated for the next experiment.

**May 23, 2022**

* HEK293 cells were seeded in a 24-well plate for the third test of Wang and colleagues’ toehold.

**May 24, 2022**

* Following the first two unsuccessful runs, several combinations were tested for the third:

|  | miR-155 | mToehold |
| --- | --- | --- |
| 1 | 800ng | 800ng |
| 2 | 400ng | 800ng |
| 3 | 800ng | 400ng |

* The three combinations were transfected to cells alongside a positive GFP control (using an existing GFP expression plasmid) and a toehold-only well (this time with 800 ng DNA).

**May 25, 2022**

* Exhibited no GFP expression in any condition but the positive GFP control.
* Results were first addressed as indicative of a problem in the toehold’s initial design and our first-cycle toeholds were designed according to this assumption.

**May 30, 2022**

* 1st cycle sequences generated by our modeling team were ordered (**Supplementary - cycles’ sequences**).

**June 7, 2022**

* Sequences arrived.

**June 14, 2022**

* Tried to run a restriction on Wang and colleagues’ miR-155 Toehold plasmid to generate a cloning backbone:

|  |  |
| --- | --- |
| ddH2O | 41 µL |
| CutSmart buffer | 5 µL |
| XhoI | 1 µL |
| BamHI | 1 µL |
| Plasmid | 2 µL |

* Ran the gel and cut it.

**June 15, 2022**

* Gel purification on the cut segment, concentration was too low.
* Tried the restriction again with similar conditions but the band in the gel was too weak for cutting.

**June 16, 2022**

* Reran the restriction in June with a different mixture (higher DNA and enzymes conc.).

|  |  |
| --- | --- |
| ddH2O | 19.5 µL |
| CutSmart buffer | 2.5 µL |
| XhoI | 1 µL |
| BamHI | 1 µL |
| Plasmid | 2 µL |

* The gel showed 2 weak bands, decided to cut the lower one as the size indicated this is the correct one.
* After Purifying the gel slices, we were only able to achieve a DNA conc. Of 13.8ng/ul.

**June 19, 2022**

* Ran Gibson using the gel output plasmid and the open GFP according to the following ratios:

|  |  |
| --- | --- |
| DDW (ul) | 0 |
| Vector (ul) | 3.6 µL |
| Open GFP fragment #1 (ul) | 0.5 |
| Mid-End GFP #2 (ul) | 0.9 |
| Gibson Assembly Mix (ul) | 5 |

* The reaction was run at 50C for 1 hour as per protocol and left overnight in 4C.

**June 20, 2022**

* Transformations with Gibson results. were run – cells underwent transformation protocol using either 5ul of the Gibson or 50ng of the cut plasmid (3.6ul cut plasmid + 1.4ul DDW) as control.

**June 21, 2022**

* Neither plate showed any colonies, leading us to think that something was wrong with the cut segment.

**June 22, 2022**

* Another attempt to run the restriction using 3ug plasmid. A cut band was observed in the gel, but when purified the Nanodrop measurements showed a yield too low to proceed.

**July 12, 2022**

* We attempted to switch to the buffer when performing the restrictions. Reran the restriction using the FastDigest buffer instead of the Cutsmart.

**July 13, 2022**

* We ran the gel and saw the proper band. However, purification gave a low concentration.
* After the revision of gel pictures, we observed a big band seen in several previous gels indicating that something might be wrong with the plasmids used.

**July 20, 2022**

* We decided to try amplifying the required segment using PCR instead and primers were ordered **(LINK)**.

**July 27, 2022**

* Primers arrived.
* PCR was attempted using the Kapa HiFi HotStart Readymix PCR kit (0.75 of each primer, 12.5ul of PCR mix, 10ul DDW and 1ul Plasmid).

|  |  |
| --- | --- |
| ddH2O | 10 µL |
| PCR mix | 2.5 µL |
| Plasmid | 1 µL |
| Primer (each) | 0.75 µL |

* The PCR was run using the following program:

|  |  |  |
| --- | --- | --- |
| 95℃ | 3 min. | X1 |
| 98℃ | 20 sec. | X35 |
| 66℃ | 15 sec | X35 |
| 72℃ | 3 min. | X35 |
| 72℃ | 1 min. | X1 |

**July 30, 2022**

* PCR results were tested on Gel, which showed a smear that existed on both the no-template control and the reaction:

**Aug 1, 2022**

* PCR was repeated with 68oc instead of 66oc annealing. The results in the gel again showed only a smear.
* Our struggle to complete the cloning process led us to consider a problem with the plasmid’s integrity in the glycerol stock. Therefore, PCR was tested on both 66℃ and 68℃ using an old plasmid that was harvested from the original starter from which the glycerol was made (13ng).
* This time, the gel for the old plasmid showed a clear band in the appropriate position’ and the ‘uncut’ plasmid.
* The bands were purified and gave 38.7 ng/ul. The resulting DNA fragment was used in a Gibson reaction according to the following table:

|  |  |
| --- | --- |
| DDW (ul) | 2.6 |
| Vector (ul) | 1.4 |
| Open Toehold fragment (ul) | 0.5 sec |
| Mid-End Fragment #2 (ul) | 0.5 |
| Gibson Assembly Mix (ul) | 5 |

* Half of the Gibson reaction was then passed through a restriction reaction with XhoI as that is supposed to get rid of any theoretical original plasmid. Incubated for 1 hr.

|  |  |
| --- | --- |
| ddH2O | 3 µL |
| FastDigest buffer | 1 µL |
| XhoI | 1 µL |
| Gibson output | 5 µL |

* The transformation was performed on 3 Amp-containing plates: 1 with 5ul of the restriction results, 1 with the remaining 5ul of uncut Gibson and 1 with 400ng of the original old plasmid (both as a positive control and since a new glycerol stock is needed).

**Aug 2, 2022**

* Colonies were observed only on the mir155-Toehold plate, indicating that something went wrong with the Gibson reaction. From this plate, 2 colonies were picked for starters (3ml LB + 3ul amp).

**Aug 3, 2022**

* we decided to repeat the Gibson reaction with increased vector-to-insert ratios:

|  |  |
| --- | --- |
| DDW (ul) | 2.6 |
| Vector (ul) | 1.4 |
| Open Toehold fragment (ul) | 0.5 sec |
| Mid-End Fragment #2 (ul) | 0.5 |
| Gibson Assembly Mix (ul) | 5 |

* Half of the Gibson reaction was then passed through a restriction reaction with XhoI as that is supposed to get rid of any theoretical original plasmid. Incubated for 1 hr.

|  | 1-to-3   1-to-4 | |
| --- | --- | --- |
| ddW (µL;L) | 2.15 | 1.7 |
| Vector (µL;L) | 1.4 | 1.4 |
| Insert#1 (µL;L) | 0.75 | 1 |
| Insert#2 (µL;L) | 0.7 | 0.9 |
| Gibson Assembly Mix (µL;L) | 5 | 5 |

* Transformation was performed with both plasmids.

**Aug 4, 2022**

* The transformation plates showed many colonies from each reaction.

**Aug 10, 2022**

* 2 colonies from each plate were extracted to LB starters.

**Aug 11, 2022**

* Half of the starters were harvested and used to make 25% Glycerol stocks, stored in -80℃.
* 1 plasmid from each plate was sent to sequencing.

**Aug 12, 2022**

* both showed the original plasmid.
* Several other colonies were sent to sequencing using the F primer to see what comes out at the beginning of the plasmid.

**Aug 14, 2022**

* PCR was rerun using less template (6.5ng vs 13ng last time) and run through gel.

**Aug 15, 2022**

* Plasmids were purified on and sent for sequencing.

**Aug 16, 2022**

* Only one starter gave the right plasmid (from the open GFP sequence) – therefore we gave it a short recovery session in the shaker before proceeding to make the glycerol.

**Aug 17, 2022**

* Gibson reactions were assembled for the other 5 plasmids according to the following table:

|  | 1.1   1.2   1.3   1.4   1.5 | | | | |
| --- | --- | --- | --- | --- | --- |
| DDW (µL;L) | 1.9 | 2.1 | 2 | 2.1 | 1.9 |
| Vector (35.7ng/µL;L) (µL;L) | 1.4 | 1.4 | 1.4 | 1.4 | 1.4 |
| Insert#1 (µL;L) | 0.8 | 0.6 | 0.7 | 0.6 | 0.8 |
| Insert#2 (µL;L) | 0.9 | 0.9 | 0.9 | 0.9 | 0.9 |
| Gibson Assembly Mix (µL;L) | 5 | 5 | 5 | 5 | 5 |

* A transfection experiment was run using the newly assembled open-toehold GFP alongside wells which included either the original miR155-Toehold plasmid or both the Toehold and trigger plasmids – it was decided to test the results by FACS after 24 hours.

**Aug 18, 2022**

* The results showed that the open GFP works, but similar results were observed in the miR155-Toehold with and without the trigger, so it is possible that the vial used was contaminated by the open plasmid.
* 5µL;L of each Gibson reaction were added to a restriction reaction:

|  |  |
| --- | --- |
| ddH2O | 3 µL |
| CutSmart buffer | 1 µL |
| XhoI | 1 µL |
| Gibson output | 5 µL |

* 1 hour at 37℃ followed by 10 minutes of inactivation at 65℃, reaction tubes were then put at -20℃.

**Aug 21, 2022**

* All 5 restriction results transformed into bacteria.

**Aug 22, 2022**

* All plates were empty of colonies.

**Aug 23, 2022**

* Another Gibson reaction was performed – since there wasn't enough PCR product for all 5 reactions, only plasmids 1-4 were made according to the following table:

|  | 1.1   1.2   1.3   1.4 | | | |
| --- | --- | --- | --- | --- |
| DDW (µL;L) | 0 | 0 | 0 | 0 |
| Vector (18.1ng/µL;L) (µL;L) | 2.8 | 2.8 | 2.8 | 2.8 |
| Insert#1 (µL;L) | 0.8 | 0.7 | 0.7 | 0.6 |
| Insert#2 (µL;L) | 1.9 | 1.9 | 1.9 | 1.9 |
| Gibson Assembly Mix (µL;L) | 5 | 5 | 5 | 5 |

* Another transformation was attempted with the Gibson results.
* HEK-293 and mouse HEPA cells were seeded in a 24-well plate.

**Aug 24, 2022**

* The plates showed many colonies.
* As an initial screen, 2 colonies were picked from each into an LB starter.
* Wang and colleagues miR-155-toehold-GFP (1) or a constitutive GFP plasmid were transfected to the cells.

**Aug 25, 2022**

* Starters were harvested for plasmids. From each plasmid, 500ng were taken into a restriction reaction.

|  |  |
| --- | --- |
| DNA | 500 µgr |
| BAmHI | 1 µL |
| XhoI | 1 µL |
| CutSmart | 2 µL |
| ddH2O | Complete to 20 µL total |

**Aug 25, 2022**

* Results were run through gel and compared to uncut plasmid extracted from the same colony.
* Successfully cloned plasmids should be cut only once. Two colonies from sequence 3 and one from sequence four seemed to be cut only once.
* HEK-293 and HEPA cells were collected, washed with PBS, and run through FACS analysis.

**Aug 28, 2022**

* The last remaining Gibson reaction (for seq. 5) was run, according to the following table:

|  |  |
| --- | --- |
| DDW (µL) | 0 |
| Vector (µL) | 3.1 |
| Open Toehold fragment (µL) | 0.8 |
| Mid-End Fragment #2 (µL) | 1.9 |
| Gibson Assembly Mix (µL) | 5 |

**Aug 29, 2022**

* The Gibson mix of seq. 5 was used for transformation.

**Aug 30, 2022**

* No colonies were observed for the seq. 5 transformation.
* The 3 seemingly correct plasmids were sent for sequencing on Aug 30th.

**Aug 31, 2022**

* Sequencing results showed that only one of the seq 3 plasmids was correct.
* 3 new colonies from the plates with seq 1,2, and 4 were collected into starters.
* The starter containing the correct seq 3 was given the night to recover.

**Sep 1, 2022**

* Glycerol stock was made from the seq 3 starter.
* The rest of the starter was harvested for DNA extraction.
* Plasmids with seq 1,2 and 4 were extracted from starters and tested by restriction with XhoI only, and the gel indicated that 2 sequences out of every triplicate might be correct.

**Sep 4, 2022**

* The plasmids that seemed correct in the gel were sent for sequencing.
* Gibson and transformation for plasmid 5 were repeated.

**Sep 5, 2022**

* Sequences were received and showed that there was at least 1 correct plasmid from each set.
* The plate for plasmid 5 showed many colonies.
* HEK293T plate was made as per protocol for testing all 4 sequences alongside the original one.
* All sequences were transfected to cells with or without the miR-155 trigger.

**Sep 6, 2022**

* The correct plasmids from the last prep were taken into a glycerol stock following an overnight recovery.
* The fluorescence of cells transfected with seq 1-4 and the original toehold was tested through FACS (see Results/mammalian toehold testing).

**Sep 7, 2022**

* 4 more colonies from the Plasmid 5 plate were picked.

**Sep 11, 2022**

* Seq 5 starters were harvested, leaving half of each.

**Sep 12, 2022**

* To test whether the GFP reduction arises specifically from miR interference, an experiment was performed in which several plasmid combinations were tested, including the open GFP plasmid and the miR, and the original plasmid alongside a Luciferase expression plasmid. All sequences were transfected to HEKs according to protocol.

**Sep 13, 2022**

* Transfection results were read using FACS (see Results/mammalian toehold testing).
* Seq 5 plasmids were tested through restriction, with the gel showing a single possibly correct plasmid.

**Sep 14, 2022**

* The putatively correct seq-5 plasmid was sent for sequencing.

**Sep 19, 2022**

* A second transfection experiment was performed for the 4 sequences and the original toehold.

**Sep 20, 2022**

* Transfection results were read using FACS (see Results/mammalian toehold testing).

**Sep 27, 2022**

* A third transfection experiment was performed for the 4 sequences, the original toehold, and the open GFP toehold. This time, cells were either transfected with the sequence alone, the sequence and its miR-155 trigger, or with the sequence and miR-21, which is not supposed to bind to the sequences.

**Sep 28, 2022**

* Transfection results were read using FACS (see Results/mammalian toehold testing).

**References:**

1. Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.

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## Toeholds Testing – Yeast

---

* Instrumentation for working with Yeast was provided courtesy of Prof. Martin Kupiec’s laboratory ([Lab website](https://mkyeastlab.wixsite.com/mklab)).
* Our Protocols can be found [here](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Lab_protocols.pdf)

**Sep 1, 2022**

* Sequences were ordered (**Supplementary - cycles’ sequences**).

**Sep 14, 2022**

* Yeast’s backbone plasmid (pRS425) was restricted using BamH1 and Hind3 restriction enzymes.

**Sep 15, 2022**

* Sequences 1-5 for the yeast experiment arrived.
* Yeasts were transformed with the sequences obtained and with cut backbone plasmids and seeded in SD-Leu plates.

**Sep 18, 2022**

* No colonies were observed on the yeast plates.
* Existing mCherry-expressing yeasts (courtesy of Kupiec lab, see above) were extracted to a YPD starter.

**Sep 19, 2022**

* The remaining sequences arrived.

**Sep , 2022**

**Sep 22, 2022**

* Another transformation attempt was done using pRS425 backbone plasmids from a different batch.
* Yeasts were transformed with a different mCherry inserted into pRS426 plasmids.

**Sep 23, 2022**

* Many colonies were detected on plates from the resulting transformed cells.

**Oct 2, 2022**

* Transformed colonies were moved to YPD to grow overnight.

**Oct 3, 2022**

* Yeast starters were washed in and transferred into 96-well plates
* Fluorescence and OD were read in Synergy H1 Microplate reader (Agilent BioTek, CA, USA).
* mCherry expression in the relevant populations was validated before GFP measured and compared between the different conditions (see Results)

---

## Parts’ Optimization

* Our Protocols can be found [here](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Lab_protocols.pdf)

---

**December 2021 – February 2022:**

- BBa\_K079050 part’s sequences (expressing GFP after exposure to UV) were optimized with the ESO program (1) and ordered.
- Bacteria were transformed with a plasmid (pSB1C3) containing the non-optimized BBa\_K079050.
- Chi.Bio (2), a robotic platform for automation of biological experiments which we wished to use both as a chemostat and a spectrophotometer, was chosen to be applied for automated evolution experiments. We attempted to measure the time it takes for the transformed bacteria to lose the fluorescent signal generated by the part when the Chi.Bio controls for constant OD to allow logistic growth and measures fluorescence.
- Due to recurrent contaminations of samples in the ChiBio device, we have decided to proceed with a manually-administered evolution experiment.

**March – April 2022:**

- Bacteria transformed with the non-optimized part were grown in selective media (LB + Chloramphenicol) and examined for fluorescence level in a plate-reader in 24-hour intervals (See Results/Parts’ optimization).
- DNA damage initiates the parts’ fluorescent activity when bacteria are applied with methyl methanesulfonate (MMS).

**May – June 2022:**

- Optimized sequences were cloned into pSB1C3 plasmids using restriction enzymes.
- After several failed cloning attempts, we revised our cloning procedure and noticed that the restriction sites in the optimized sequences were too short for the enzymes chosen (EcoRI and PstI), therefore we tried to use other restriction enzymes (XbaI and SpeI), for which restriction was successful.
- A manual evolution experiment was done as described above, with fluorescent signals measured daily (see Results/Parts’ optimization).

**References:**

1. Menuhin-Gruman, I., Arbel, M., Amitay, N., Sionov, K., Naki, D., Katzir, I., ... & Tuller, T. (2021). Evolutionary Stability Optimizer (ESO): A Novel Approach to Identify and Avoid Mutational Hotspots in DNA Sequences While Maintaining High Expression Levels. ACS Synthetic Biology, 11(3), 1142-1151.
2. Steel, H., Habgood, R., Kelly, C. L., & Papachristodoulou, A. (2020). In situ characterisation and manipulation of biological systems with Chi. Bio. PLoS biology, 18(7), e3000794.

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* [Introduction](#introduction)
* [First Experiment](#first)
* [Second Experiment](#second)
* [Third Experiment](#third)
* [Yeast Experiment](#yeast)
* [Model Engineering Cycles](#model)
* [References](#ref)

## Introduction

Our work in the project was conducted in a cycle of four parts: Design, Build, Test and Learn.

![](https://static.igem.wiki/teams/4219/wiki/engineering/eng-suc.png)

The engineering cycle

**Design**- Generating Toehold switch sequences using our Model.

**Build** - Inserting the generated sequence to GFP expressing plasmid.

**Test** - Determining the toehold switch effectiveness in vitro.

**Learn** - implementing new insights into the Models and the cycle repeats.

The results obtained in the Test part led us to new conclusions and the needed improvements implemented in the Model. Then we could repeat design with the improved Model, Build, Test and learn again.

In general, we can divide our work into 4 experiments, all of which undergo at least one engineering cycle. The first was reproducing a former study in eukaryotes with miRNA as the trigger. This experiment, being the first one, underwent the largest number of engineering cycles, as we tried to calibrate our systems. Eventually, the lab results led us to critical conclusions. Therefore, we changed the Model and added new features. For example, we added the prevention of stop codons and limited the sequence to single start codons which have to be in the reading frame.

The second experiment included an improved sequence, still based on the former study but with our optimizations by our Model. The results of the second experiment showed that we succeeded in improving the performance of the toehold and therefore was our first engineering success. It also led us to realize that miRNA might be too short for eukaryotic switches, which led us to designing the next experiment for mRNA triggers. The difficulties and length of experiments with mammalian cells also led us to design an experiment in yeast, which will be discussed later.

The third experiment was designing sequences from scratch using mRNA as a trigger. The lab results showed that we are getting high ON concentration (protein abundance in the presence of the mRNA) but also high OFF concentration (protein abundance in the absence of the mRNA). Therefore, we decided to design different structures, for example one that contains two hairpins instead of one in order to lower the OFF ratio. In parallel, as mentioned earlier, we learned that working with mammalian cells can be time consuming and cause many difficulties, so we designed an experiment in yeast. In this experiment we decided to check a few different conditions for validation of our models parameters.

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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**Toehold Results**
**Parts Results**

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## POC results – In-vivo Toehold Screening

As a major part of our project was aimed at developing a platform for generating effective toehold elements for specific eukaryotic translation, we had to provide evidence for our program-generated sequences’ efficiency. As not much formerly published work exists on toehold in eukaryotes, we had little to instruct us in choosing between different toehold characteristics. We thus utilized the Toehold tryouts as an opportunity to examine possible features of eukaryotic toehold design, with the hope of implementing the conclusions in the program and retesting the outputs in later cycles. Previous studies in the field have not focused on endogenously expressed triggers; thus, we chose not to focus on endogenously expressed triggers, as we figured this challenge should be addressed only after an effective toehold structure is attained for eukaryotes. We chose to start with Wang and colleagues’ original toehold as a point of comparison of our optimization results [1] as other works chose to use alternative structures to the classical toehold structures for specific translation in eukaryotes [2].

To effectively examine Wang et al. Toehold's performances compared to ours, we tried to test the sequences in a system as similar to the one used in their work as we could attain (Figure 1). Plasmids with different miR-155-reactive toeholds sequences positioned 5’ to GFP reporter genes were examined and were compared to the miR-155-GFP plasmids used by Wang and colleagues (Figure 2), which were sent to us by the courtesy of the author [1]. Our initial budget constraints led us to use miRNA expression plasmids as triggers instead of the miRNA mimics used in the article, which might have some impact on toeholds performances. All sequences were tested in HEK-293 cells, which were seeded in 24-well plates and transfected using Lipofectamine 2000 24 hours later. Each toehold-GFP plasmid was transfected twice into cells, either alone or with the miRNA expression plasmid. Its GFP expression and fold change between the two conditions were measured via Fluorescence Activated Flow Cytometry (FACS) and compared to Wang et al.’s plasmid and to a constitutive GFP expression plasmid (figure 1).

![](https://static.igem.wiki/teams/4219/wiki/results/results1.png)

**Figure 1:** outline of toehold testing in mammalian cell lines

Sequences are cloned into plasmids and transfected to HEK-293 cells seeded in 24-well plates 24 hours prior. Toehold sequences are either transfected alone or together with a plasmid expressing the trigger 155-miRNA.

---

### Results from Wang et al.’s sequence suggest a negative effect of miRNA on expression

Four versions of the miR-155-toehold-GFP were created using our program (Figure 2; **[Supplementary – cycles’ sequences](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Supplementary___cycles__sequences.xlsx)**): (1) A sequence designed such that translation would only start after the stem-loop, meaning that the Kozak sequence and start codon are found after the toehold; (2) a sequence conceptually similar to the design in Wang and colleagues, meaning that a start codon and a Kozak sequence exist both at the middle of the stem-loop and at the beginning of the GFP’s open reading frame (ORF) and that the GFP’s ORF is not aligned with the first start codon; (3) a sequence generated according to the ‘classical’ toehold design in bacteria [3], meaning that an AUG and the Kozak sequence exist only inside the stem-loop; and (4) a sequence similar to the former but with an additional step in the design pipeline that prevents the formation of pseudoknots surrounding the toehold structure (see Model page for a better description of sequences’ design pipeline).

![](https://static.igem.wiki/teams/4219/wiki/results/results2.png)

**Figure 2:** Toehold sequences tried in the 1st cycle

Colors represent different sequence elements: Orange – trigger-binding area; Red – Kozak sequence and start codon; Green – beginning of the GFP’s ORF. **a.** The sequence taken from Wang and colleague [1], Kozak and AUG are present both inside the loop and adjacent to the ORF. **b.** Four sequences generated by our program, with the Kozak and start codon found after (1) or inside (3, 4) the toehold stem-loop, or both (2). Sequence 4 was added with a pseudoknot prevention step.

To minimize additional intervening factors in the comparison between the toeholds, we decided to use Wang and colleagues’ expression plasmid (pCDNA3(-)) as a backbone to clone our sequences into. We chose to construct our plasmids from 3 parts, with the 2nd half of the GFP ORF inserted independently from the rest of the sequence, allowing us to use a ‘universal’ insert in a manner that fitted our logistical constraints. We used the PCR reaction to open and amplify only the backbone from Wang et al.’s plasmid and then constructed the new plasmid via Gibson assembly (Figure 3a). As Gibson’s success rates proved low, and to minimize costly and time-consuming sequencings, we screened plasmids from colonies of bacteria transformed with DNA from the Gibson reaction through restriction of a sample with XhoI (which cuts only inside the original toehold but not in any of our inserts) and running it through gel electrophoresis, marking uncut wells as suspected successful reactions and sending only these colonies for sequencing (figure 3b).

![](https://static.igem.wiki/teams/4219/wiki/results/results3a.png)
![](https://static.igem.wiki/teams/4219/wiki/results/results3b.png)

**Figure 3:** Toehold’s cloning procedure

**a.** Scheme of our cloning procedure, the backbone of the plasmid from Wang et al.[1] was amplified using PCR. 155-Toehold-GFP sequences were constructed with Gibson assembly from a constant middle-end GFP part and a variable 155-Toehold part. **b.** Gel electrophoresis of DNA samples extracted from colonies transformed with Gibson reaction output. Samples were either loaded directly (lower panel) or after restriction with XhoI (upper panel, see Lab notebook). Unsuccessful reactions are cut and present one lighter bend in the upper panel. Cut samples were excluded from further analyses.

When examining Wang et al.’s toehold’s performances, we noticed that in contrast with the results reported when using miRNA mimetics [1], our run of the miR-155 Toehold provided consistently lower expressions when co-transfected with the trigger miRNA expression plasmid (Figure 4a). To examine if the reduction in expressing population is resulting from the excessive load of plasmid co-transfection or from a direct and specific effect of the miRNA interacting with the toeholds, we compared its activity alone and with the 155-miRNA to its activity when co-transfected with a miRNA-21 plasmid, which is not expected to interact with the toeholds as it doesn’t bare the relevant trigger sequence. We could not see a large difference in reduction caused when co-transfecting with miRNA-155 or miRNA-21, but were able to repeat this procedure only once, and therefore prefer to apply caution when interpreting this result (Figure 5b). We also compared the different conditions for a constitutive GFP plasmid instead of the toehold, which is not supposed to interact with either miRNA. When examining the effect of co-transfection with miRNA-155 on this plasmid, it is apparent that expression is in fact not reduced in the double-transfected population, suggesting a specific negative effect of the miRNA-toehold interactions on translation (Figure 4b).

![](https://static.igem.wiki/teams/4219/wiki/results/4-1-res.png)
![](https://static.igem.wiki/teams/4219/wiki/results/4-2-res.png)

**Figure 4:** FACS results for Wang et al.’s miR-155 toehold

Co-transfection with miRNA-155 specifically decreases GFP expression in miR-155-Toehold-GFP. **a.** GFP is expressed in cells transfected with the miR-155 toehold construct (two right panels) but is lower when miRNA-155 expression plasmid is present (right). **b.** Open (constitutively expressing) GFP plasmid is unaffected by the existence of miRNA-155 (low expression is present overall). Scatterplots of FACS measurements were taken from live, single cells. The threshold for GFP expression (X-axis) was set according to the untreated cells (see Methods).

---

### 1st cycle’s sequence 1 had the highest fold-change of all sequences examined

As in Wang and colleagues' toehold, two of our four sequences exhibited consistently lower expression rates when transfected with the trigger. Results were also worse in these sequences when compared to expression with the non-binding miRNA-21 sequence instead of the trigger miRNA-155, but only one repetition was performed with this miRNA (Figure 5b). Of the other sequences, sequence 1, in which the start codon and the Kozak sequence are found only after the stem-loop element, consistently exhibited similar or better expression rates when transfected with the trigger than when transfected alone (Figure 5). Sequence 1 thus presented a seemingly higher fold-change than all other toeholds, although insignificantly according to the student’s t-test (However we believe further repetition of the testing would solve this issue). Overall expression was higher in the sequences having only the upstream start codon (seq. 4, 3), however in a non-specific manner.

![](https://static.igem.wiki/teams/4219/wiki/results/results5.png)

**Figure 5:** FACS results for our program-generated sequences

**a.**Summed FACS results (n = 3 repetitions) of the transfection experiments’ shown as fold-change ![](https://static.igem.wiki/teams/4219/wiki/results/equation.png). When co-transfected with the trigger, sequence 1 consistently presents results higher or similar to the ones measured when transfected alone. **b.** FACS scatterplots showing GFP fluorescence in cells transfected with Wang et al.’s[1] Toehold-GFP or with our 1sr cycle sequences: alone (upper row), together with the trigger miRNA-155 expression plasmid (middle row), or with a non-binding miRNA-21 expression plasmid (lower row). FACS measurements were taken from live, single cells (see Methods).

---

### mRNA could be used as triggers for toeholds in eukaryotes

One of our project’s main goals was to use windows inside mRNAs as triggers for eukaryotic toeholds. Despite mRNAs being formerly used to trigger toeholds in prokaryotes3 and in IRES-based eukaryotic toehold-like structures [2], we could not find any work that uses the classical toehold structure with triggers in mRNA. The ability to use sequences in mRNAs could highly expand the ensemble of triggers to choose from and thus expand the applicability of these structures for targeting to different cell populations. Working with miRNA-triggered toeholds in the 1st cycle further strengthened our decision to move to mRNA triggers as we found it difficult to assess our success in expressing the trigger plasmid, because of the limited tools for miRNA transcripts identification and our limited resources and time. Specifically, with mRNA, we could use a fluorescent trigger to quickly overcome this issue. As results from the 1st cycle suggested a specific effect of the miRNA-toehold interaction, decreasing protein expression, we wanted to check if this effect stems from the miRNA’s nature and could be avoided when using mRNA instead.

Therefore, we decided to further examine our toehold sequences using mCherry-mRNA as a trigger. We did this cycle with S. cerevisiae instead of the mammalian cell line, which allowed us to screen sequences in higher throughput and also to verify our software’s applicability to eukaryotes other than mammalian cells. We examined sequences with the two best-targeting designs from the first cycle, sequences 1 (Kozak downstream to the stem-loop) and 4 (Kozak inside the loop), but this time we were able to use our computational assessment method to choose the few highest-ranked sequences for each design option (**[Supplementary – cycles’ sequences](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Supplementary___cycles__sequences.xlsx)**), Metrics used are specified in the Model section). Sequences in this cycle are therefore ranked according to the probability of a successful interaction to occur. We used Gap Repair to clone our different Toehold-GFP sequences into a constitutive expression vector (pRS425) and compared GFP levels to those in yeast also transfected with mCherry expression plasmids (cloned inside pRS426).

![](https://static.igem.wiki/teams/4219/wiki/results/results6.png)

**Figure 6:** Fluorescence results for the second cycle in yeast

Fluorescence was measured in a plate reader and normalized to OD (600 nm). Results are shown as fold-change ![](https://static.igem.wiki/teams/4219/wiki/results/equation2.png). Asterisks on each bar represent statistical significance of the difference between expression with or without trigger sequence. Sequences 1 and 6, predicted to be the optimally active sequences in each category (**[Supplementary – cycles’ sequences](https://gitlab.igem.org/2022/tau-israel/-/raw/main/supplementary/Supplementary___cycles__sequences.xlsx)**) show significantly higher fold-changes compared to other toeholds in each category. \* p-value < 0.05, \*\* p-value < 0.01, \*\*\* p-value < 0.001, \*\*\*\* p-value < 0.0001.

Interestingly, the two highest-ranked sequences in both categories (Kozak up and inside) also exhibited the highest fold increase when administered with the trigger. These sequences, Y1 (Kozak after the stem-loop) and Y6 (Kozak inside) were the only ones constitutively higher expressed when transformed together with mCherry plasmid (Figure 6). These results strengthen our assessment method’s validity and suggest a successful Toehold triggering by mRNA, which had not yet been reported in eukaryotes. Of special interest is the relative success of toeholds with the Kozak and start codon situated after the stem loop, a structure different from the one commonly used in bacteria. Based on these results and other advancements in our software, we constructed a 3rd cycle of sequences, but were unable to complete its testing by the Wikis’ closure deadline. We also checked expression levels without the trigger normalized to the yeast’s autofluorescence (measured in WT yeast under the same conditions). And the fluorescence of the toehold-GFP sequences compared to a constitutively expressed GFP sequence. All toehold-GFP sequences’ fluorescence was shown to be insignificantly different from autofluorescence when administered without the trigger sequence (student’s t-test), meaning that all sequences exhibited low leakiness of expression in non-target cells (Figure 7a). When administered together with the mCherry trigger, both Y1 and Y6 exhibited similar expression rates to the constitutive GFP sequence (student’s t-test), which, together with the low expression without the trigger accounts for a highly effective toehold activity (Figure 7b).

![](https://static.igem.wiki/teams/4219/wiki/results/7-res.png)

**Figure 7:** mRNA-triggered toeholds in yeast exhibit low leakiness without the trigger and effective expression with it

**a.** Fluorescent from yeast transformed with toeholds only (off-state) divided by autofluorescence of non-treated wild-type yeast. All sequences exhibited insignificant differences between off-state expression autofluorescence, assessed by student’s t-test. **b.** Fluorescence in yeast applied both toehold-GFP sequences and the trigger mCherry sequence, divided by fluorescence in yeasts with a constitutively expressed GFP plasmid. \* p-value < 0.05, \*\* p-value < 0.01, \*\*\* p-value < 0.001, \*\*\*\* p-value < 0.0001. Student’s t-test.

To conclude the results of our toeholds testing, we were able to use the successive cycles to further develop our software and adapt it according to the results (see Project Engineering for additional adaptations made). We were also able to show improved activity of one of our toeholds over the previously reported eukaryotic toehold. In the yeast experiments, we were able to exhibit - for the first time, to our knowledge - effective toehold activity of an mRNA-triggered toehold in eukaryotes. Interestingly, the structure which worked best both in mammals and in yeast was the one of which start codon and Kozak sequence was downstream to the stem-loop element, a design different from the ones previously reported. Based on these findings and on our toehold assessment method, we designed another cycle of toeholds to be tested in mammalian cells, for which results are still pending.

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### References

1. Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.
2. Zhao, E. M., Mao, A. S., de Puig, H., Zhang, K., Tippens, N. D., Tan, X., ... & Collins, J. J. (2022). RNA-responsive elements for eukaryotic translational control. Nature Biotechnology, 40(4), 539-545.
3. Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925-939.

---

## Parts’ Improvement

Genetic stability is a fundamental theme of synthetic biology. Genetic constructs, fulfilling a specific destination in a synthetic cycle, must preserve their functionality over long periods of time, avoiding mutations that lead to the loss of the gene’s functionality. However, such preservation requires energy production, resulting in a reduction in fitness. In order to form a stable-functioning construct with an intact fitness value, novel tools of synthetic biology need to be brought into action. The Evolutionary Stability Optimizer (ESO) software is a computational tool developed by the iGEM TAU 2020 team, which is directed at automatically generating genetic constructs with preserved functionality and a minimal loss of fitness [1].

For our project’s part-optimization component, we utilized the ESO program to improve the Evolutionary stability of an existing part stored on the iGEM registry, BBa\_K079050. We optimized it using the software in two fashions, a milder one and a fully-optimized one (for more details about ESO, visit the [ESO website](https://www.cs.tau.ac.il/~tamirtul/ESO)). To experimentally examine the effect of this optimization, we compared the evolutionary stability of the standard iGEM part in comparison with the evolutionary stability of the same part after optimization, by looking at its fluorescent signal over time. We tried to show that the optimized part (in both versions) reserves its functionality for longer times than the non-optimized one, so we designed the experiment in a way that enabled us to follow the process of losing functionality.

![](https://static.igem.wiki/teams/4219/wiki/results/001-res.png)

**Figure 1:** Expression levels (fluorescence) of non-optimized and optimized parts

*E. coli* transformed with the different parts were grown in restrictive media, split to allow continuous growth, and their fluorescence levels were measured daily. The results are shown in Figure 1. Optimization by the ESO software stabilized expression levels of the construct: expression levels of the non-optimized part were significantly lower than those of the mild optimized one (p-value = 4\*10-3), student’s t-test) and those of the full optimized one (p-value = 9.7\*10-7); as mentioned, the evolutionary stability (figure 2) has been measured as the time it took the construct to lose its fluorescence (and thus, its functionality). It was evident that the original, non-optimized construct lost its fluorescent signal faster than both the optimized constructs (while the mild optimized construct lost its fluorescence faster than the fully optimized one).

![](https://static.igem.wiki/teams/4219/wiki/results/002-res.png)

**Figure 2:** GFP fluorescence over time

GFP fluorescence levels over time (excit. 484; emmit. 510), optimized and non-optimized parts. Green - original non-optimized GFP; Red - minor optimization and Blue - full optimization. Each one performed as triplicates, marked by dotted, dashed, and solid lines. The non-optimized part lost its functionality in a short time - a significant decrease in fluorescence just after 2 days and no fluorescence after 3 days. Both minor-optimization and fully optimized parts took longer to lose their functionality - a decrease in fluorescence after 3-4 days and a loss of functionality in 5 or more days.

---

We would like to report about another results of an optimization process taken out by the ESO tool that has been performed over an existing part of the iGEM parts registry - BBa\_J04421 (an eCFP coding device, containing a promoter, an RBS, a coding sequence and a terminator). The same procedures were taken while dealing with that part, in order to detect its expression levels and evolutionary stability. The results are depicted in figure 3, both the evolutionary stability and the expression levels, represented by the GFP fluorescence values of the different versions of the part (non-optimized/merely optimized/fully optimized) in day 1, the initial measurements timepoint. It could be witnessed regarding that part as well that the ESO tool has optimized its evolutionary stability and its expression levels.

![](https://static.igem.wiki/teams/4219/wiki/results/respart2.jpeg)

**Figure 3:** eCFP fluorescence over time

eCFP fluorescence levels over time (excit. 434; emmit. 477), optimized and non-optimized parts. Solid - original non-optimized eCFP; dashed - minor optimization and dotted - full optimization. Each one performed as triplicates, marked by different lines. The non-optimized part lost its functionality in a short time - a significant decrease in fluorescence just after 3 days and no fluorescence after 4 days. Both minor-optimization and fully optimized parts took longer to lose their functionality - a slight decrease in fluorescence after 4 days and a loss of functionality in 5 or more days.

To conclude, the successful application of the ESO software to optimize the BBa\_K079050 part, our results support the possibility that the ESO tool bears the ability to improve evolutionary stability and expression levels of genetic constructs. This ability is an important one considering the need to preserve both the functionality and fitness of genetic constructs in various Syn. Bio. applications.

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### References

1. (1) Menuhin-Gruman, I., Arbel, M., Amitay, N., Sionov, K., Naki, D., Katzir, I., ... & Tuller, T. (2021). Evolutionary Stability Optimizer (ESO): A Novel Approach to Identify and Avoid Mutational Hotspots in DNA Sequences While Maintaining High Expression Levels. ACS Synthetic Biology, 11(3), 1142-1151.

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