iGEM 2020 — Team Evry_Paris-Saclay

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# Team:Evry Paris-Saclay/Notebook

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![](https://static.igem.org/mediawiki/2020/7/73/T--Evry_Paris-Saclay--2020--medal--or.png)
![](https://static.igem.org/mediawiki/2020/b/bf/T--Evry_Paris-Saclay--Logo-Rosewood_txt_light.png)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:Evry Paris-Saclay/Engineering

![](https://static.igem.org/mediawiki/2020/9/94/T--Evry_Paris-Saclay--Logo-iGEM-Evry-2020-squared-96.png)
![](https://static.igem.org/mediawiki/2020/7/73/T--Evry_Paris-Saclay--2020--medal--or.png)
![](https://static.igem.org/mediawiki/2020/b/bf/T--Evry_Paris-Saclay--Logo-Rosewood_txt_light.png)

# Engineering Success

The rosewood biosensor is based on toehold switches (Figure 1) which are RNA–based devices containing a ribosome binding site (RBS) and an ATG start codon embedded in a hairpin structure that blocks translation initiation [[1]](#ref1). The hairpin can be unfolded upon binding of a trigger RNA thereby exposing the RBS and permitting translation of the reporter protein. The binding between the trigger and the switch is dictated by RNA-RNA interactions between the complementary base pairs. In the past, such biosensors have been employed to identify relevant biological material and mutations specific to diseases like cancer [[2]](#ref2) and Zika virus [[3]](#ref3) and more recently the SARS-CoV-2 virus [[4,5]](#ref5).
![Toehold switch](https://static.igem.org/mediawiki/2020/7/7a/T--Evry_Paris-Saclay--toehold-switch.png)
\*\*Figure 1.\*\* Toehold switches principle (schematic inspired from iGEM Athens 2018).
The Rosewood biosensor project was inspired by the engineering framework of Synthetic Biology: Design, Build, Test and Learn [DBTL]. We can clearly identify two complete DBTL cycles during the project duration that were invaluable for our team’s progress.
## Rosewood DBTL Cycle #1
### Design
The first iteration of the DBTL cycle used the toehold switches predicted by the CUHK tool [[6]](#ref6). This was done with two purposes in mind. First, we wanted to get used to the experimental protocols relating toehold switches construction and testing while our own model was under construction. More importantly, we wanted to build these toehold switches with the goal of comparing our model against the previously published software [[6]](#ref6) (Check our ["Model"](https://2020.igem.org/Team:Evry\_Paris-Saclay/Model) page for a more detailed description).
### Build
The toehold switch sensors (Table 1) were assembled by Golden Gate in the low copy plasmid pSB3T5. For this, fragments containing the T7 promoter followed by different toehold switches and RBS were synthesized with flanking type IIS restriction sites, and then interested into one of the acceptor vectors [BBa\_K3453101](http://parts.igem.org/Part%3ABBa_K3453101%20), [BBa\_K3453102](http://parts.igem.org/Part%3ABBa_K3453102%20) or [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103%20) in which Golden Gates adapters are present upstream of sfGFP-LVAtag with either BsaI, BsmBI or BbsI sites, respectively.
The trigger sequences (Table 2) were placed under the control of the T7 promoter and followed by the strong SBa\_000587 synthetic terminator and the resulting transcriptional units were synthesized and assembled in the high copy plasmid pSB1C3.
\*\*Table 1.\*\* Rosewood toehold switches sensors designed by the CUHK tool [[6]](#ref6).

| Label | Toehold Switch Part Number | sfGFP Expression Cassettes’ Part Number |
| --- | --- | --- |
| Rosewood DmMatK Toehold Switch 1.1 | [BBa\_K3453011](http://parts.igem.org/Part%3ABBa_K3453011%20) | [BBa\_K3453111](http://parts.igem.org/Part%3ABBa_K3453111%20) |
| Rosewood DmMatK Toehold Switch 1.3 | [BBa\_K3453012](http://parts.igem.org/Part%3ABBa_K3453012%20) | [BBa\_K3453112](http://parts.igem.org/Part%3ABBa_K3453112%20) || Rosewood DmMatK Toehold Switch 1.4 | [BBa\_K3453013](http://parts.igem.org/Part%3ABBa_K3453013%20) | [BBa\_K3453113](http://parts.igem.org/Part%3ABBa_K3453113%20) || Rosewood DmRbcL Toehold Switch 1.1 | [BBa\_K3453031](http://parts.igem.org/Part%3ABBa_K3453031%20) | [BBa\_K3453131](http://parts.igem.org/Part%3ABBa_K3453131%20) |
| Rosewood DmRbcL Toehold Switch 1.2 | [BBa\_K3453032](http://parts.igem.org/Part%3ABBa_K3453032%20) | [BBa\_K3453132](http://parts.igem.org/Part%3ABBa_K3453132%20) |
| Rosewood DmRbcL Toehold Switch 1.3 | [BBa\_K3453033](http://parts.igem.org/Part%3ABBa_K3453033%20) | [BBa\_K3453133](http://parts.igem.org/Part%3ABBa_K3453133%20) |
| Rosewood DmTrnL-UAA Toehold Switch 1.1 | [BBa\_K3453051](http://parts.igem.org/Part%3ABBa_K3453051%20) | [BBa\_K3453151](http://parts.igem.org/Part%3ABBa_K3453151%20) |
| Rosewood DmTrnL-UAA Toehold Switch 1.2 | [BBa\_K3453052](http://parts.igem.org/Part%3ABBa_K3453052%20) | [BBa\_K3453152](http://parts.igem.org/Part%3ABBa_K3453152%20) |
| Rosewood DmTrnL-UAA Toehold Switch 1.3 | [BBa\_K3453053](http://parts.igem.org/Part%3ABBa_K3453053%20) | [BBa\_K3453153](http://parts.igem.org/Part%3ABBa_K3453153%20) |

\*\*Table 2.\*\* Rosewood triggers designed by the CUHK tool [[6]](#ref6).

| Label | Trigger Part Number | Trigger Transcriptional Unit Part Number |
| --- | --- | --- |
| Rosewood DmMatK Trigger 1.1 | [BBa\_K3453021](http://parts.igem.org/Part%3ABBa_K3453021%20) | [BBa\_K3453121](http://parts.igem.org/Part%3ABBa_K3453121%20) |
| Rosewood DmMatK Trigger 1.3 | [BBa\_K3453022](http://parts.igem.org/Part%3ABBa_K3453022%20) | [BBa\_K3453122](http://parts.igem.org/Part%3ABBa_K3453122%20) |
| Rosewood DmMatK Trigger 1.4 | [BBa\_K3453023](http://parts.igem.org/Part%3ABBa_K3453023%20) | [BBa\_K3453123](http://parts.igem.org/Part%3ABBa_K3453123%20) |
| Rosewood DmRbcL Trigger 1.1 | [BBa\_K3453041](http://parts.igem.org/Part%3ABBa_K3453041%20) | [BBa\_K3453141](http://parts.igem.org/Part%3ABBa_K3453141%20) |
| Rosewood DmRbcL Trigger 1.2 | [BBa\_K3453042](http://parts.igem.org/Part%3ABBa_K3453042%20) | [BBa\_K3453142](http://parts.igem.org/Part%3ABBa_K3453142%20) |
| Rosewood DmRbcL Trigger 1.3 | [BBa\_K3453043](http://parts.igem.org/Part%3ABBa_K3453043%20) | [BBa\_K3453143](http://parts.igem.org/Part%3ABBa_K3453143%20) |
| Rosewood DmTrnL-UAA Trigger 1.1 | [BBa\_K3453061](http://parts.igem.org/Part%3ABBa_K3453061%20) | [BBa\_K3453161](http://parts.igem.org/Part%3ABBa_K3453161%20) |
| Rosewood DmTrnL-UAA Trigger 1.2 | [BBa\_K3453062](http://parts.igem.org/Part%3ABBa_K3453062%20) | [BBa\_K3453162](http://parts.igem.org/Part%3ABBa_K3453162%20) |
| Rosewood DmTrnL-UAA Trigger 1.3 | [BBa\_K3453063](http://parts.igem.org/Part%3ABBa_K3453063%20) | [BBa\_K3453163](http://parts.igem.org/Part%3ABBa_K3453163%20) |

#### Positive and negative controls
As positive controls for the above described sfGFP expression cassettes, we designed two parts, all harbouring the sfGFP-LVAtag under the control of T7 promoter and the strong SBa\_000587 synthetic terminator, but with different RBSes :
- In [BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104%20), the sfGFP-LVAtag was equipped with a custom made RBS ([BBa\_K2675017](http://parts.igem.org/Part%3ABBa_K2675017%20)).
- In [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105%20), the sfGFP-LVAtag was equipped with the synthetic stem-loop containing an RBS designed by Pardee \*et al.\* [[3]](#ref3) to be part of a standardized toehold switch ([BBa\_K3453005](http://parts.igem.org/Part%3ABBa_K3453005%20)).
As negative control, we used [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103%20) in which no promoter and no RBS are present upstream of sfGFP-LVAtag.
### Test
For toehold switch characterization we decided to use *E. coli* BL21 Star™(DE3) cells (Thermo Fisher Scientific), genotype F- \*ompT hsd\*SB (rB-mB-) \*gal dcm rne131\* (DE3). As all BL21(DE3) \*E. coli\* strains, these cells contain the T7 RNA polymerase under control of the lacUV5 promoter and thus require IPTG to induce expression. The particularity of BL21 Star™(DE3) cells is that they contain a truncated version of the RNaseE gene (\*rne131\*) that leads to reduced level of mRNA degradation and thus increased RNA stability.
For fluorescence measurements, \*E. coli\* cells containing switch and trigger plasmids were first grown overnight in 96-deep-well plates containing 1 mL of LB medium supplemented with 5 µg/mL tetracycline and 17.5 µg/mL chloramphenicol, then diluted by 40x into similar media. Upon reaching early log-phase, cells were further diluted 20x in 100 µL of LB medium supplemented with 5 µg/mL tetracycline, 17.5 µg/mL chloramphenicol and 10 µM IPTG in an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). The plate was incubated at 37°C at 200 rpm and the sfGFP fluorescence (λexcitation 483 nm and λemission 530 nm) and OD600 were measured every 10 min for 24 hours in a CLARIOstar (BMGLabtech) plate reader.
Fluorescence values were normalised by OD600 and, using the calibration curves presented on the ["Measurement"](https://2020.igem.org/Team:Evry\_Paris-Saclay/Measurement) page of this wiki, we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle.
### Learn
#### Controls
We first measured a pair of positive and negative controls. The results presented in Figure 2 show that the sfGFP expression was readily detected in the positive controls and that the two RBSes ([BBa\_K2675017](http://parts.igem.org/Part%3ABBa_K2675017%20) and [BBa\_K3453005](http://parts.igem.org/Part%3ABBa_K3453005%20)) have comparable strength. As expected, no fluorescence was detected in the negative controls as either the sfGFP gene was not present or the promoter and the RBS were absent.
![ParticleStdCurve Clario](https://static.igem.org/mediawiki/2020/e/eb/T--Evry_Paris-Saclay--p22-p23-p24_Fluo-OD600-means.png)
\*\*Figure 2.\*\* \*In vivo\* characterization of sfGFP expression by \*E. coli\* BL21 Star™(DE3) cells harbouring the positive controls ([BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104%20%20%20) and [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105%20%20%20)) and the negative controls [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103%20%20) (no promoter, no RBS) or an empty pSB3T5 vector. Cells were co-transformed with an empty pSB1C3 backbone in order to rule out any antibiotic influence on the \*E. coli\* growth and metabolism. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.
#### Fluorescence results
When tested against their cognate triggers (Figure 3), 7 of the 9 first-generation switches showed fluorescence with a mean comparable to that of the positive controls, except for two switches (DmMatK 1.3 and DmRbcL 1.3). When tested in the no trigger condition, three of the first-generation toehold switches (DmRbcL 1.2, DmTrnL-UAA 1.1 and DmTrnL 1.2) exhibited fluorescence higher than 50% of the average of positive controls which we define as leaky behaviour.
![ParticleStdCurve Clario](https://static.igem.org/mediawiki/2020/a/a2/T--Evry_Paris-Saclay--MEFL-Particle_p35-p51.png)
\*\*Figure 3.\*\* \*In vivo\* characterization of sfGFP expression by \*E. coli\* BL21 Star™(DE3) cells harbouring the 1st generation of rosewood toehold switches (Table 1) and triggers (Table 2). The negative controls have been performed with an empty pSB3T5, pSB1C3 (no trigger) and [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103%20%20) (no promoter, no RBS) and the positive controls with [BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104%20%20) and [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105%20%20). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.
#### ON/OFF Ratio
The positive controls showed on average 320 times the fluorescence of the negative controls confirming the validity of the experimental setup. Of the 9 first-generation toehold switches tested, DmMatK 1.1 exhibits an ON/OFF ratio greater than 100, DmMatK 1.4 shows an ON/OFF level close to 30 and DmMatK 1.3, DmRbcL 1.1 and DmTrnL 1.3 display at least a level greater than 5 (Figure 4).
![ParticleStdCurve Clario](https://static.igem.org/mediawiki/2020/c/ce/T--Evry_Paris-Saclay--Fold-change_p35-p51.png)
\*\*Figure 4.\*\* MEFL / Particle fold changes of the 1st generation of rosewood toehold switches (Table 1) in the presence of the rosewood triggers (Table 2) compared to the MEFL / Particle value in the absence of the trigger.
#### Crosstalk
To measure orthogonality of the toehold switches, we performed additional crosstalk experiments. In these experiments, we tested the expression of the reporter gene when the trigger of a different toehold switch for the same gene was added inside the cell. Only three of the 9 first-generation toehold switches showed a higher fluorescence measurement in the presence of their non-cognate trigger or even in the absence of any trigger: DmRbcL 1.2, DmTrnL-UAA 1.1 and 1.2. The other 6 toehold switches generated a fluorescent response only in the presence of a trigger containing the corresponding binding site. It should be noted that the triggers designed with the CUHK tool [[6]](#ref6) are 120 nt long and some contain binding sites for several switches.
#### Full trigger
In addition, we tested the toehold switches in the presence of the respective full known fragments of \*Dalbergia maritima\* genes, which we refer to as the full triggers. The MEFL / Particle values were generally lower which indicates lower levels of switch - trigger binding most probably due to steric constrains.
#### Toehold switches showed a dynamic range that leverages the full expression capability of the cassette
The high dynamic range of fluorescence outputs without and with their cognate triggers showed us that we had enough expression change to easily distinguish between the negative and the positive results. We expected that the measurements obtained from the toehold switches will show a reduced dynamic range between the ranges shown by the control. However, as shown in Figure 3, we obtained positive results that surpassed the fluorescence of the positive controls suggesting that we leveraged the full capacity of the expression cassette.
#### Low leaky expression is key for obtaining high-performance toehold switches
We obtained a range of ON/OFF ratios expanding over two orders of magnitude (Figure 4). The highest performer switch, DmMatK 1.1, showed an ON/OFF ratio of 130, on the same order of performance as some of the best switches reported in literature. The positive signal of the switch is approximately 40% higher to the signal obtained for the positive controls, but not the highest positive signal that we obtained on the dataset. In contrast, the fluorescence measured in the absence of the trigger was the lowest among all the first-generation switches. For comparison, the switch DmMatK 1.4 had a positive signal also comparable to that of positive controls but its negative output was almost 5 times higher than that of DmMatK 1.1. Consequently, the ON/OFF ratio of DmMatK 1.4 is markedly reduced. Thus, large ON/OFF ratios are based primarily on very low levels of leakage expression found in the absence of the trigger.
#### Some genes targets seemed more easily detectable
On this very small sample size, the three toehold switches for DmMatK showed favorable behavior albeit with very different ON/OFF ratios. On the contrary, we obtained only one favorable switch for DmTrnL-UAA. It is tempting to suggest that DmMatK is an easier target in terms of the secondary structures that can be achieved to detect its sequence. However, given our small sample size and several other parameters that may have reduced DmTrnL-UAA toehold switches performance we would have to more rigorously test this hypothesis.
#### Crosstalk is detectable but at desirable levels
In contrast to other sensing purposes where a set of toehold switches that are highly orthogonal is desirable, the ability to detect triggers that are slightly off from the cognate trigger desired means that we will be able to detect triggers that have some mutations in their sequences.
The extent of this crosstalk needs to be measured as we clearly want to avoid false positives in our sensing. A possible approach for this would be to create a spectrum of triggers from negative controls to full triggers that measures the activation curve of the switches in relationship to the similarity to the cognate trigger sequence.
#### Efficacy score of the CUHK model was a poor predictor of toehold switch performance
As can be seen in Table 3, the efficacy score weakly predicted fluorescent measurements of the toehold switches. In particular, the two designs with highest expected performance \*in silico\* did not produce functional toehold switches. In addition, the poorest design in terms of efficacy turned out to be the toehold switch with the second highest fold change ratio. These results suggest that we could have obtained better results using toehold switches with lower predicted ranks and confirm the challenging nature of predicting RNA secondary structure dynamics \*in silico\*.
\*\*Table 3.\*\* Efficacy scores of the Rosewood toehold switches sensors designed by the CUHK tool [[6]](#ref6) vs their ranking according to fold change ratio measured \*in vivo\* (Figure 4). It should be noted that the last digit of the toehold name is the rank predicted by the CUHK tool.

| Label | Efficacy score *in silico* | Rank *in vivo* |
| --- | --- | --- |
| Rosewood DmMatK Toehold Switch 1.1 | 98 | 1 |
| Rosewood DmMatK Toehold Switch 1.3 | 55 | 5 || Rosewood DmMatK Toehold Switch 1.4 | 44 | 3 || Rosewood DmRbcL Toehold Switch 1.1 | 35 | 6 |
| Rosewood DmRbcL Toehold Switch 1.2 | 32 | - |
| Rosewood DmRbcL Toehold Switch 1.3 | 15 | 2 |
| Rosewood DmTrnL-UAA Toehold Switch 1.1 | 115 | - |
| Rosewood DmTrnL-UAA Toehold Switch 1.1 | 110 | - |
| Rosewood DmTrnL-UAA Toehold Switch 1.3 | 110 | 4 |

## Rosewood DBTL Cycle #2
### Design
The second iteration of the DBTL cycle used the toehold switches predicted by our own in-house pipeline. A script written in Python uses the \*multitubedesign\* function of the NuPACK suite, a software package for RNA secondary structure prediction, for inverse RNA folding [[7]](#ref7). It takes a target sequence as well as a desired toehold-switch structure in its on- and off-state as an input and outputs the most promising sequence. By performing a single nucleotide sliding window analysis of our target genes, we generated a large list of putative switches. Those were then ranked and chosen according to several metrics: (i) the predicted Translation initiation rate (TIR), calculated with the RBS calculator [[8]](#ref8), (ii) the Hamming distances between the predicted and the desired structures, and (iii) the minimum free energies of the different switch domains using the NuPACK mfe function. Of ~1000 switches for each gene, three switches were chosen for each gene to be further tested (Check our [“Model”](https://2020.igem.org/Team:Evry\_Paris-Saclay/Model) page for a more detailed description).
### Build
We built the second-generation toehold switches (Table 4) and triggers (Table 5) following the procedures described in the first DBTL cycle.
\*\*Table 4.\*\* Rosewood toehold switches sensors designed using our own in-house pipeline.

| Label | Toehold Switch Part Number | sfGFP Expression Cassettes’ Part Number |
| --- | --- | --- |
| Rosewood DmMatK Toehold Switch 2.1 | [BBa\_K3453014](http://parts.igem.org/Part%3ABBa_K3453014%20) | [BBa\_K3453114](http://parts.igem.org/Part%3ABBa_K3453114%20) |
| Rosewood DmMatK Toehold Switch 2.2 | [BBa\_K3453015](http://parts.igem.org/Part%3ABBa_K3453015%20) | [BBa\_K3453115](http://parts.igem.org/Part%3ABBa_K3453115%20) || Rosewood DmMatK Toehold Switch 2.3 | [BBa\_K3453016](http://parts.igem.org/Part%3ABBa_K3453016%20) | [BBa\_K3453116](http://parts.igem.org/Part%3ABBa_K3453116%20) || Rosewood DmRbcL Toehold Switch 2.1 | [BBa\_K3453034](http://parts.igem.org/Part%3ABBa_K3453034%20) | [BBa\_K3453134](http://parts.igem.org/Part%3ABBa_K3453134%20) |
| Rosewood DmRbcL Toehold Switch 2.2 | [BBa\_K3453035](http://parts.igem.org/Part%3ABBa_K3453035%20) | [BBa\_K3453135](http://parts.igem.org/Part%3ABBa_K3453135%20) |
| Rosewood DmRbcL Toehold Switch 2.3 | [BBa\_K3453036](http://parts.igem.org/Part%3ABBa_K3453036%20) | [BBa\_K3453136](http://parts.igem.org/Part%3ABBa_K3453136%20) |
| Rosewood DmTrnL-UAA Toehold Switch 2.1 | [BBa\_K3453054](http://parts.igem.org/Part%3ABBa_K3453054%20) | [BBa\_K3453154](http://parts.igem.org/Part%3ABBa_K3453154%20) |
| Rosewood DmTrnL-UAA Toehold Switch 2.2 | [BBa\_K3453055](http://parts.igem.org/Part%3ABBa_K3453055%20) | [BBa\_K3453155](http://parts.igem.org/Part%3ABBa_K3453155%20) |
| Rosewood DmTrnL-UAA Toehold Switch 2.3 | [BBa\_K3453056](http://parts.igem.org/Part%3ABBa_K3453056%20) | [BBa\_K3453156](http://parts.igem.org/Part%3ABBa_K3453156%20) |

\*\*Table 5.\*\* Rosewood triggers designed using our own in-house pipeline.

| Label | Trigger Part Number | Trigger Transcriptional Unit Part Number |
| --- | --- | --- |
| Rosewood DmMatK Trigger 2.1 | [BBa\_K3453024](http://parts.igem.org/Part%3ABBa_K3453024%20) | [BBa\_K3453124](http://parts.igem.org/Part%3ABBa_K3453124%20) |
| Rosewood DmMatK Trigger 2.2 | [BBa\_K3453025](http://parts.igem.org/Part%3ABBa_K3453025%20) | [BBa\_K3453125](http://parts.igem.org/Part%3ABBa_K3453125%20) || Rosewood DmMatK Trigger 2.3 | [BBa\_K3453026](http://parts.igem.org/Part%3ABBa_K3453026%20) | [BBa\_K3453126](http://parts.igem.org/Part%3ABBa_K3453126%20) || Rosewood DmRbcL Trigger 2.1 | [BBa\_K3453044](http://parts.igem.org/Part%3ABBa_K3453044%20) | [BBa\_K3453144](http://parts.igem.org/Part%3ABBa_K3453144%20) |
| Rosewood DmRbcL Trigger 2.2 | [BBa\_K3453045](http://parts.igem.org/Part%3ABBa_K3453045%20) | [BBa\_K3453145](http://parts.igem.org/Part%3ABBa_K3453145%20) |
| Rosewood DmRbcL Trigger 2.3 | [BBa\_K3453046](http://parts.igem.org/Part%3ABBa_K3453046%20) | [BBa\_K3453146](http://parts.igem.org/Part%3ABBa_K3453146%20) |
| Rosewood DmTrnL-UAA Trigger 2.1 | [BBa\_K3453064](http://parts.igem.org/Part%3ABBa_K3453064%20) | [BBa\_K3453164](http://parts.igem.org/Part%3ABBa_K3453164%20) |
| Rosewood DmTrnL-UAA Trigger 2.2 | [BBa\_K3453065](http://parts.igem.org/Part%3ABBa_K3453065%20) | [BBa\_K3453165](http://parts.igem.org/Part%3ABBa_K3453165%20) |
| Rosewood DmTrnL-UAA Trigger 2.3 | [BBa\_K3453066](http://parts.igem.org/Part%3ABBa_K3453066%20) | [BBa\_K3453166](http://parts.igem.org/Part%3ABBa_K3453166%20) |

### Test
We tested the second-generation toehold switches following the procedures described in the first DBTL cycle.
### Learn
#### Fluorescence results
The nine toehold-switches were tested with and without their cognate trigger, the full-length gene and, if there was sequence similarity, against additional triggers (Figure 5). When tested in the no trigger condition, three second-generation toehold switches (DmMatK 2.2, DmMatK 2.3 and DmTrnL 2.1) showed leaky behavior as they produced similar high fluorescence in absence of their cognate trigger.
![Toehold switch](https://static.igem.org/mediawiki/2020/a/a0/T--Evry_Paris-Saclay--MEFL-Particle_p67-p75.png )
\*\*Figure 5.\*\* \*In vivo\* characterization of sfGFP expression by \*E. coli\* BL21 Star™(DE3) cells harbouring the 2nd generation of rosewood toehold switches (Table 4) and triggers (Tables 2 and 5). The negative controls have been performed with an empty pSB3T5, pSB1C3 (no trigger) and [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103%20) (no promoter, no RBS) and the positive controls with [BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104%20) and [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105%20). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.
#### ON/OFF Ratio
ON/OFF ratios of the nine second-generation toehold switches were calculated by dividing MEFL / Particle values of switch with trigger and switch without trigger (Figure 6). DmRbcL 2.2 and DmTrnL-UAA 2.2 show very high fold-change with values exceeding 40 and 20 respectively. DmMatK 2.1 ranks third with a ratio of almost 10, while the other six switches don’t exceed a fold change of five.
![Toehold switch](https://static.igem.org/mediawiki/2020/3/3d/T--Evry_Paris-Saclay--Fold-change_p67-p75.png)
\*\*Figure 6.\*\* MEFL / Particle fold changes of the 2nd generation of rosewood toehold switches (Table 4) in the presence of the rosewood triggers (Table 5) compared to the MEFL / Particle value in the absence of the trigger.
#### Crosstalk
Broad crosstalk tests were not performed for the second-generation switches, though five of the nine switches were also tested with triggers of other switches, if sequence overlap occurred.
#### The sensing performance of the second-generation switches is impaired by high values in negative scenarios
Even though the toehold switches showed high expression in the presence of their cognate trigger, we report high fluorescence values in their negative scenarios. This effect caused a reduction in most of the ON/OFF ratios and greatly impacted the sensing capabilities. Several reasons could explain this leakiness, among them:
1. Switch sequences cannot fold into a hairpin structure stable enough to resist ribosome binding and stem unzipping.
2. Endogenous RNAs from \*E. coli\* bind and unwind stem structure
A criterion we use to prevent choosing leaky switches, is TIR fold-change and OFF-TIR values predicted by the RBS calculator [[8]](#ref8). TIR itself is put together by different variables including Shine-Dalgarno similarity, ribosome binding free energy penalty, potential for ribosome sliding and others that rely on RNA secondary structure and RNA biophysics predictions.
To improve leakiness, we aim to both adjust the cutoffs for TIR and the other metrics and, to generate a more diverse library and have a broader range of possibilities, predict switches of multiple architectures, that vary in domain length (toehold, stem, loop) and predefined sequence and structural constraints.
#### Unlike that suggested by the DBTL Cycle #1 data, our gene targets do not seem to influence toehold switch performance
In contrast to behaviour seen in DBTL Cycle #1, DmTrnL-UAA in Cycle #2 proved to be a gene easier to detect with the second-generation toehold switches, and, on the contrary, DmMatK showed leaky behaviour. This suggests that the difficulties encountered in the two cycles are not particular to the sequence of the gene tested but rather more likely due to the toehold design methods.
## Conclusions
We have successfully designed, built, tested and analysed 18 rosewood toehold switches, half of which were are able to act as toehold switches and (i) efficiently repress the downstream reporter gene expression in the absence of the cognate trigger and (ii) release the translation inhibition in the presence of the cognate trigger. Moreover, we obtained several functional toehold switches for each of the three \*Dalbergia maritima\* genes tested: MatK, RbcL and TrnL-UAA, and 6 of them were further tested in a cell-free experimental setup as a Proof of Concept experiment for the implementation of our Rosewood detection tool (Check our ["Proof of Concept"](https://2020.igem.org/Team:Evry\_Paris-Saclay/Proof\_Of\_Concept) page for a more detailed description).
## References
[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159: 925–939.
[2] Hong F, Ma D, Wu K, Mina LA, Luiten RC, Liu Y, Yan H, Green AA. Precise and programmable detection of mutations using ultraspecific riboregulators. Cell (2020) 180: 1018-1032.e16.
[3] Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, Daringer NM, Bosch I, Dudley DM, O’Connor DH, Gehrke L, Collins JJ. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell (2016) 165: 1255–1266.
[4] Boettner B. Harvard Wyss Institute’s nasal swab and toehold switch technologies licensed to Agile Biodetection to facilitate SARS-CoV-2 diagnostic efforts. Wyss Institute2020 Oct 5.
[5] Koksaldi IC, Ahan RE, Kose S, Haciosmanoglu N, Kehribar ES, Gungen MA, Ozkul A, Seker UOS. SARS-CoV-2 detection with de novo designed synthetic riboregulators. medRxiv (2020): 2020.07.28.20164004.
[6] To AC-Y, Chu DH-T, Wang AR, Li FC-Y, Chiu AW-O, Gao DY, Choi CHJ, Kong S-K, Chan T-F, Chan K-M, Yip KY. A comprehensive web tool for toehold switch design. Bioinformatics (2018) 34: 2862–2864.
[7] Zadeh JN, Steenberg CD, Bois JS, Wolfe BR, Pierce MB, Khan AR, Dirks RM, Pierce NA. NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry (2011) 32: 170–173.
[8] Salis HM. The ribosome binding site calculator. Methods in Enzymology (2011) 498: 19–42.

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# Team:Evry Paris-Saclay/Experiments

![](https://static.igem.org/mediawiki/2020/9/94/T--Evry_Paris-Saclay--Logo-iGEM-Evry-2020-squared-96.png)
![](https://static.igem.org/mediawiki/2020/7/73/T--Evry_Paris-Saclay--2020--medal--or.png)
![](https://static.igem.org/mediawiki/2020/b/bf/T--Evry_Paris-Saclay--Logo-Rosewood_txt_light.png)

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Evry Paris-Saclay/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)