iGEM 2022 — Team evry-paris-saclay

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## Engineering

Our project Electricia coli is based on the use of a genetically modified *Escherichia coli* as a biosensor
that
emits an electrical signal when detecting a specific cancer biomarker: the PANTR1 long non-coding RNA (lncRNA).
Our Electricia coli biosensor is based on the use of a toehold switch **[1]** (Figure 1). This RNA-based device
is
composed of several parts which are essential for the efficient functioning of our biosensor:

* a

  Trigger Binding Site (TBS)

  which is a sequence
  complementary to the RNA
  target
  to be detected
* a

  Ribosome Binding Site (RBS)

  which allows the start of the
  translation only
  when
  the toehold switch is unfolded
* a

  start codon AUG

  which constitutes the start of the
  translation
* a

  linker

  which constitutes a bridge between the Start codon
  and our gene of
  interest
* a

  gene of interest

  that is translated into a
  protein only when the
  hairpin is unfolded and the RBS
  exposed

![](https://static.igem.wiki/teams/4432/wiki/modeling/image1.png)

**Figure 1.** Principle of the toehold switch used for Electricia coli (inspired from iGEM Evry
Paris-Saclay 2020).

The functioning comes as follows:
The binding between the RNA biomarker and the toehold switch unfolds the hairpin structure. The RBS becomes accessible
now so that the translation can take place, and the initially repressed gene of interest can then be translated into a
protein. In the Electricia coli project, the translated protein plays a role in the production of electrons. By using
an MCF, electrons are captured and converted into an electrical signal, which is recorded by an embedded system.

We were inspired by previous such biosensors designed to specifically identify various biological nucleic acid
sequences originating from viruses **[2–7]**, trees, **[8]** or cancer related mutations **[9]**, and applied
the DBTL (Design,
Build, Test, Learn) framework, a hallmark of synthetic biology, in order to develop high performing toehold switches
as biosensors that detect the PANTR1 lncRNA.

## Electricia coli “Design – Build – Test – Learn” Cycle

### Design

Our aim was to give *E. coli* the ability to detect the lncRNA PANTR1. To do so, we have to design some toehold
switches
with computational approaches, and test *in vivo* the ones with the best performance. We use superfolder green
fluorescent protein (sfGFP) as the gene of interest for easy screening of protein expression.

The prediction of the toehold switches was made with the tool developed by the Hong Kong-CUHK iGEM 2017 team
**[10]** for each of the four versions of the PANTR1 lncRNA. We choose to adopt the previously optimized
architecture of the toehold switch sensors for Zika virus detection **[2]** that proved its efficiency on other
toehold switches too **[8,11]**. This architecture comprises a custom Loop sequence (GGACUUUAGAACAGAGGAGAUAAAGAUG)
and a custom Linker sequence (AACCUGGCGGCAGCGCAAAAG), which we specified as parameters in the CUHK web tool
**[10]**.

A total of 396 distinct toehold switches were thus designed for the
PANTR1 lncRNA, some of them being common to several versions of PANTR1:

* 368 for the PANTR1 v1 (GenBank Acc. N° NR\_037883)
* 38 for the PANTR1 v2 (GenBank Acc. N° NR\_131233)
* 35 for the PANTR1 v3 (GenBank Acc. N° NR\_131234)
* 44 for the PANTR1 v4 (GenBank Acc. N° NR\_131235)

The full list and and their parameters are available as an [excel
file](https://gitlab.igem.org/2022/evry-paris-saclay/-/blob/main/data/Result_CUHK_PANTR1.xlsx).

The CUHK tool has an embedded RNAfold algorithm that predicts the minimum free energy (MFE) of trigger, switch,
switch-domains, and trigger-switch complex, and a ranking function that attributes an efficacy score to each toehold
switch based on a linear regression model trained on 181 switches and their ON/OFF rates.

For PANTR1, we observed that one quarter of the designed toehold switches had predicted efficacy scores greater than 1
(Figure 2), and that the majority of them were specific to PANTR1 v1, which is statistically expected as the length of
its sequence is greater than those of PANTR1 v2, v3 and v4.

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic2-pantr1-efficacyscore.png)

**Figure 2.** Efficacy scores (ON/OFF rates) calculated by the CUHK model of the all switches
designed for PANTR1.

Among all the designed switches, we selected the top 3 best ranked and their cognate triggers to characterize them
*in
vivo* in *E. coli*, for a total of 8 switches and 10 triggers (Table 1) by taking into account the
overlapping
sequences
between the four versions of PANTR1.

These 8 switches, designated hereafter by numbers, have slightly different secondary structures (Figure 3) and
thermodynamic characteristics (Table 2). The minimum free energy (MFE) of the trigger-switch heterodimer is at least
25.1 and up to 43.2 kcal/mol lower than the total MFE of trigger and switch monomers. This value termed MFE difference
in the CUHK model output represents the Gibbs free energy change needed for the RNA:RNA interaction to proceed. These
8 switches also have a high difference between the MFE of trigger-switch dimer and either of the trigger-trigger and
the switch-switch dimers suggesting that the activated switch conformation is favored thermodynamically.
Following these calculations, we expect that the switch n°8 will have beneficial result slightly higher than switch
n°1 and that the switch n°7 will have lesser performance with any of the 3 cognate triggers. Comparing efficacy scores
between genes, it is expected that the toehold switches designed to identify PANTR1 v1 lncRNA will have a much higher
performance compared to those designed for the other three versions.

**Table 1.** Top 3 best ranked toehold switches designed by the CUHK pipeline for the four PANTR1
variants. This set of 8 toehold switches was later synthesized and evaluated *in vivo* in *E. coli.*

| Label | Targeted gene and rank number | | | |
| --- | --- | --- | --- | --- |
| PANTR1 v1 | PANTR1 v2 | PANTR1 v3 | PANTR1 v4 |
| n°1 | Rank 1 | - | - | - |
| n°2 | Rank 2 | - | - | - |
| n°3 | Rank 3 | - | - | - |
| n°4 | Rank 23 | Rank 1 | Rank 1 | Rank 2 |
| n°5 | Rank 52 | Rank 2 | Rank 2 | Rank 4 |
| n°6 | Rank 58 | Rank 3 | Rank 4 | Rank 5 |
| n°7 | Rank 54 (trigger n°7c) | - | Rank 3 (trigger n°7a) | Rank 3 (trigger n°7b) |
| n°8 | - | - | - | Rank 1 |

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic3-pantr1-dot-brackets.png)

**Figure 3.** Secondary structures of the top toehold switches represented in dot-bracket notation
coloured to match the different segments in Figure 1.

**Table 2.** Minimum free energy (MFE) of trigger, switch, switch-domains and trigger-switch
complex and efficacy scores estimated by the CUHK pipeline for the 8 PANTR1 toehold switches selected for *in
vivo*
characterization. Values are expressed in kcal/mol.

| Label | Minimum Free Energy (MFE) | | | | | | | Efficacy Score |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| RBS-Linker | Switch | Trigger | Trigger-Switch Dimer | Switch-Switch Dimer | Trigger-Trigger Dimer | MFE Difference |
| n°1 | -6.4 | -18.3 | -18.3 | -77.5 | -42.0 | -44.2 | 40.9 | 182.24 |
| n°2 | -5.8 | -28.0 | -19.3 | -84.3 | -60.0 | -50.2 | 37.0 | 140.84 |
| n°3 | -6.5 | -21.2 | -25.2 | -82.5 | -47.8 | -60.5 | 36.1 | 133.38 |
| n°4 | -6.4 | -26.2 | -27.9 | -85.9 | -59.1 | -64.5 | 31.8 | 74.68 |
| n°5 | -7.0 | -32.4 | -22.3 | -90.6 | -69.9 | -51.7 | 35.9 | 30.67 |
| n°6 | -4.9 | -36.1 | -21.6 | -92.0 | -77.3 | -48.4 | 34.3 | 25.61 |
| n°7a | -4.8 | -23.6 | -23.2 | -71.9 | -51.2 | -52.6 | 25.1 | 28.65 |
| n°7b | -4.8 | -23.6 | -22.8 | -72.8 | -51.2 | -50.2 | 26.4 | 37.62 |
| n°7c | -4.8 | -23.6 | -21.4 | -70.1 | -51.2 | -51.9 | 25.1 | 28.65 |
| n°8 | -6.6 | -28.5 | -28.3 | -100.0 | -63.5 | -64.7 | 43.2 | 79.61 |

### Build

The 8 selected toehold switches sensors were assembled by Golden Gate technique in the low copy plasmid pSB3T5. For
this, we
designed and
synthesized fragments containing the T7 promoter followed by the different toehold switches, the RBS Loop and the
Linker sequences, with flanking
type IIS restriction sites (BsaI) and inserted them into [BBa\_K3453101](http://parts.igem.org/Part%3ABBa_K3453101), which contains a Golden Gate adapter with BsaI
sites upstream of the sfGFP-LVAtag.

The 10 selected trigger sequences were placed under the control of the T7 promoter and followed by the strong
SBa\_000587
synthetic terminator. The resulting transcriptional units were then synthesized and assembled in the high copy
plasmid pSB1C3.

The list and sequences of PANTR1 toehold switches and triggers is accessible through our [Basic Parts](https://2022.igem.wiki/evry-paris-saclay/parts),
[Composite Parts](https://2022.igem.wiki/evry-paris-saclay/parts-composite) and [Parts Collection](https://2022.igem.wiki/evry-paris-saclay/parts-collection)
sections.

### Test

For toehold switch characterization, cells of *E. coli* BL21 Star™(DE3) (Thermo Fisher Scientific) were used.
This
strain was chosen, first of all because it contains inserted in its genome the T7 RNA polymerase gene, required for
the transcription initiation of all PANTR1 toehold switches sensors and triggers. It is under the control of the
lacUV5 promoter and requires IPTG to induce expression. And second because the it has the particularity to contain
a truncated version of the RNAseE gene (*rne131*), which reduces the level of RNA degradation and thus increases
RNA
stability.

*E. coli* cells containing both the toehold switch sensor and the trigger plasmids were grown overnight at 37
°C at
200 rpm in 96-deep-well plates with 1 mL of LB, in which was added 5 µg/mL tetracycline and 17.5 µg/mL
chloramphenicol. The cells were then diluted by 40 times in the same media and after 4 hours incubation at 37°C at
200 rpm, they were further diluted by 20 times in media containing also 10 µM IPTG in an opaque wall 96-well
polystryrene microplate (COSTAR 96, Corning). The plate was then incubated at 37°C at 200 rpm and the sfGFP
fluorescence (λexcitation 488 nm and λemission 530 nm) and optical density at 600nm
(OD600) were measured every 10 minutes for 24 hours, in a CLARIOstar (BMGLabtech)
plate reader.

Fluorescence values were normalized by OD600. Furthermore, the arbitrary units have been converted into
Molecules
of Equivalent FLuorescein (MEFL) / particle (more details about the calibration curves are available on the [Measurement](https://2022.igem.wiki/evry-paris-saclay/measurement) page
on this wiki).

### Learn

#### Controls showed expected results

The results we obtained with the pairs of control (Figure 4) showed that the sfGFP expression was detected in the
positive controls (custom made RBS and standard toehold switch RBS stem-loop) while there was no sfGFP expression
detected in the negative controls (empty backbone and no promoter + no RBS), as expected.

#### Three toehold switches significantly expressed sfGFP only in presence of PANTR1

Fluorescence results represented in Figure 4 reveals that among all the switches tested, 3 of them (n°5, 6 and 8)
showed no fluorescence (or a negligible one) when the trigger was missing and a Fluorescence/OD600nm
comparable to the
positive controls when the trigger is present. This difference (i.e. the ON/OFF ratio) reaches a value of 102 for the
toehold switch n°6 which is thus our best RNA sensor, followed by switches n°8 and n°5 with fold changes of 40 and 15
respectively (Figure 5).

Three other switches (n°1, 3, 4) were able to express sfGFP to a level comparable to that of the positive controls in
the presence of their cognate triggers, but, in the absence of the trigger, leakiness was observed. Their ON/OFF
ratios (1.1, 1.5 and 2.4) are poor and were not further considered.
Switches n°2 and 7 are not functional. No fluorescence is observed in any condition. The sfGFP expression is tightly
controlled and the presence of the trigger does not allow the hairpin to unfold.

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic4-pantr1-all-meflperparticle.png)

**Figure 4.** *In vivo* characterization of sfGFP expression by *E. coli* BL21
Star™(DE3)
cells
carrying the PANTR1 toehold switches and triggers. The negative controls have been performed with an empty pSB3T5
and [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103) (no promoter, no RBS), and
pSB1C3 (no
trigger, “-” symbol) for every Switch. The positive controls have been performed with [BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104) (custom made RBS) and [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105) (standard toehold switch RBS stem-loop). The data
and error bars are the mean and standard deviation of at least three measurements on independent biological
replicates.

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic5-pantr1-all-foldchange.png)

**Figure 5.** MEFL/Particle fold changes of the PANTR1 toehold switches in the presence of their
cognate triggers compared to the MEFL/Particle value in the absence of the trigger.

#### Crosstalk

Additional crosstalk experiments were carried out to evaluate our 3 functional toehold switches behavior in the
presence of other triggers.

We thus tested switches n°5, 6 and 8 in the presence of the other 9 non-cognate PANTR1 triggers. Results presented in
Figure 6 and 7 revealed that Switch n°8 showed significant fluorescence only with its cognate trigger n°8, while
Switch n°5 and n°7 crosstalk:

* Switch n°5 showed a significant fluorescence with cognate trigger n°5 and non-cognate trigger n°6
* Switch n°6 showed fluorescence with trigger n°6 and a low fluorescence with non-cognate trigger n°5.

These results are not unexpected as switches n°5 and n°6 have an overlap of 31/35 nucleotides.

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic6-pantr1-568-meflperparticle.png)

**Figure 6.** *In vivo* characterization of sfGFP expression by *E. coli* BL21
Star™(DE3)
cells
carrying the PANTR1 toehold switches n°5, 6 or 8 and all other triggers. The negative controls have been performed
with an empty pSB3T5, [BBa\_K3453103](http://parts.igem.org/Part%3ABBa_K3453103) (no
promoter, no RBS) and
pSB1C3 (no trigger), and the positive controls with [BBa\_K3453104](http://parts.igem.org/Part%3ABBa_K3453104) and [BBa\_K3453105](http://parts.igem.org/Part%3ABBa_K3453105). The data and error bars are the mean and standard
deviation of at least three measurements on independent biological replicates.

![](https://static.igem.wiki/teams/4432/wiki/engineering/pic7-pantr1-568-foldchange.png)

**Figure 7.** MEFL/Particle fold changes of the PANTR1 toehold switches n°5, 6 or 8 in the
presence of each of all the other triggers compared to the MEFL/Particle value in the absence of the trigger.

#### Efficacy scores of the CUHK model were different from *in vivo* toehold switches performance

The efficacy scores for PANTR1 toehold switches estimated by the CUHK model showed a different rank of efficacy than
the one observed *in vivo*: Switch n°6 showed the best results *in vivo*, while Switch n°1 showed the best
efficacy
score *in silico* (Table 3). The efficacy score of the CUHK model alone was thus a poor predictor of *in
vivo* efficacy of
PANTR1 switches, in our case, as already described in the literature **[8]**.

**Table 3.** Efficacy scores of PANTR1 toehold switches designed by the CUHK tool (Figure 2) vs
their ranking according to fold change ratio measured *in vivo* (Figure 5).

| Label | Efficacy score *in silico* | Efficacy rank *in vivo* |
| --- | --- | --- |
| PANTR1 Switch n°1 + Trigger 1 | 182.24 | 6 |
| PANTR1 Switch n°2 + Trigger 2 | 140.84 | 7 |
| PANTR1 Switch n°3 + Trigger 3 | 133.38 | 5 |
| PANTR1 Switch n°4 + Trigger 4 | 74.68 | 4 |
| PANTR1 Switch n°5 + Trigger 5 | 30.67 | 3 |
| PANTR1 Switch n°6 + Trigger 6 | 25.61 | 1 |
| PANTR1 Switch n°7 + Trigger 7a | 28.65 | 8 |
| PANTR1 Switch n°7 + Trigger 7b | 37.62 | 10 |
| PANTR1 Switch n°7 + Trigger 7c | 28.65 | 9 |
| PANTR1 Switch n°8 + Trigger 8 | 79.61 | 2 |

## Conclusion

We have successfully designed, built, tested and analyzed 8 PANTR1 lncRNA specific toehold switches in *E. coli*.
One of
them (switch n°6) was strongly expressing the downstream sfGFP gene reporter in the presence of its cognate PANTR1
trigger and strongly repressing its translation in the absence of the trigger. It’s ON/OFF ratio of 101 highlights its
performance and places it among the best switches described in the literature.

Two others (switches n°5 and 8) were expressing sfGFP too, with a lower but very decent ON/OFF ratio.

These results can let us conclude the efficiency of 3 PANTR1 specific toehold switches in *E. coli* to detect
the
trigger and express a gene of interest in response, and thus the ability of our Electricia coli to act as a biosensor
of PANTR1 lncRNA.

## References

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eaat5105.

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