iGEM 2022 — Team insa-lyon1

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![Shocking](https://static.igem.wiki/teams/4239/wiki/mascotte/mascotte-coucou.png)

![Shocking](https://static.igem.wiki/teams/4239/wiki/mascotte/mascotte-ampoule.png)

## 16

### Oct

Here you can find the calendar of our project and the notebook of our experiments. Follow us in every step of our work!

## 2021

## October

### October 16th

DNA synthesis with Genecust voucher, sponsored by Assystem.

## November

### November 16th

Team meeting: final decision about the topic.

### November 19th

Team meeting: reorganization of the team in sub-teams, planning of the first experiments.

## December

### December 1st

Team logo first proposition.

### December 2nd

Team Meeting.
 Finalization of the brochure for presentation/sponsoring.
 Start of the budget.

### December 3rd

Start of sponsoring research.

### December 14th

Team meeting.

## 2022

## January

### January 10th

Meeting with inov3PT, first sponsor.

## February

### February 3rd

Team meeting.
 Reflexion about team mascot.
 Discussion about findings as regards image analysis/modelisation.

### February 12th

Team meeting.
Reflexion about wiki design.

## March

### March 16th

Creation of our crowdfunding page in collaboration with the "Fondation INSA".

### March 22th

Forum at the biosciences department.

### March 23th

Forum at the biosciences department.

### March 27th

First textiles design.
 Reception of first donation from FSDIE.

### March 28th

Team meeting.
 iGEM reglementation overview.
 Start of wiki design.
 Dry lab: Functional graphical interface.
 Meeting with PI.

## April

### April 4th

CROUS donation.

### April 11th

Meeting with the "Fondation INSA" to discuss our crowdfunding strategy.

### April 19th

ABIL donation.

### April 20th

Team Meeting.
Start of the board game.

## May

### May 5th

Meeting with human practice supervisors: Brainstorming, integration to the team.

### May 11th

First call with a farmer president of the Union of agricultural unions in Aisnes.

### May 13th

Meeting with the "Fondation INSA" to discuss communication strategy.

### May 15th

Team Meeting.
BioRender licence.
Snapgene licence.

### May 17th

Crowdfunding platform launching.

### May 21st

Team Meeting.

### May 23rd

Official opening of our crowdfunding platform + APIE subvention presentation.

### May 24th

Meeting with sponsor Promega.

### May 31st

Encounter with Auriane Vicente, graphic designer.

## June

### June 2nd

Team meeting.
Obtention of 20kb DNA from IDT sponsor.
Dry lab: statement of work and planning of the tasks.

### June 13th

Call with iGEM Marseille for IDT.

### June 14th

Meeting with the "Fondation INSA" to discuss how to sollicit new industrial partners.

### June 15th

Interview Loick Le Meur (UNPT).
Team meeting: organization of vulgarization and education.

### June 16th

Bayer visit and encounter with researchers.
Team meeting.

### June 19th

iGEM Human Practice Workshop.

### June 20th

Submission of the impact grant.
Catch-up team meeting.

### June 21st

Interlab webinar.

### June 22nd

Catch-up team meeting.
NEB kits received.

### June 23rd

Call with Canada team.

### June 24th

Safety form due.

### June 27th

Biomérieux meeting.
Call with mentor.

## July

### July 2nd

Team meeting for promotional video.

### July 4th

Start of summer wet lab.
Call with Biomérieux.

### July 5th

Team meeting.

### July 7th

iGEM teams meetup France.

### July 8th

iGEM teams meetup France.

### July 9th

iGEM teams meetup France.
Team meeting: medals and video.

### July 11th

inov3PT director meeting.
Webinar: modeling.

### July 15th

Track selection.
Roster freeze.

### July 19th

Peer review for MSP journal articles.

### July 20th

First meeting with the school director to organize a scientific activity with children.

### July 21st

Brainstorming for promotional video.

### July 25th

Deadline article MSP.

### July 26th

Writing of promotion video script.

### July 28th

Team meeting.
Intervention within Maison de l'Enfance Henri Wallon with 6-11 yo children in a summer camp.

### July 31st

Official closing of our crowdfunding platform.

## August

### August 1st

iGEM impact grant news!

### August 4th

Team meeting.

### August 15th

Giant Jamboree and part of team member tickets purchased.

### August 19th

English + German voice-over recorded for promotional video.

### August 22nd

Testing day (judging form, parts page, etc).

### August 23rd

Team meeting.

### August 31st

Team interview by GenScript.

## September

### September 1st

Promotional video submission.

### September 2nd

Final invoice for Jamboree Paris housing.

### September 10th

Team-building week-end.
Team meeting.

### September 11th

Team-building week-end.

### September 12th

Sweat-shirts and polos order.

### September 13th

Meeting with Bayer at the LifeHub.

### September 14th

SynBio Symposium in Paris.
Lauch of the HP consumer survey.
Chicory infecton.

### September 15th

Project safety form submitted + offical team roster.
Meeting with school principals to organise interventions.
 Start of Enzymatic experiments.
Team-building restaurant.

### September 16th

Presentation of the project in front of the third-year students INSA Lyon.
Meeting with mechanical student for hardware modelling.

### September 17th

Video call with Kubii.

### September 19th

Jamboree housing in Paris fully paid.

### September 21st

Meeting with our school director and teachers to present the project

### September 22nd

Biomérieux visit to our lab.

### September 23rd

Interview SNIPT.
Meeting with iGEM Patras.
Modelling meeting with Hubert Charles

### September 25th

Team meeting organized by the KOREA-HS team.

### September 30th

Seminar with researchers from the MAP laboratory.

## October

### October 6th

Intervention within a middle school.

### October 8th

Intervention within Ebullisciences.

### October 12th

Judging form + team wiki + registry.

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![banière](https://static.igem.wiki/teams/4239/wiki/schemas-communs/baniere-gauche.png)

# ENGINEERING

![banière](https://static.igem.wiki/teams/4239/wiki/schemas-communs/baniere-droite.png)

- [**Creating Biobricks**](#eng_1)
- [**Determination of optimal conditions**](#eng_2)
- [**Implementation in *D. solani***](#eng_3)
- [**Mathematical Model**](#eng_4)
- [**Enzymatic Kinetics**](#eng_5)

FIAT LUX’s goal was to modify and adapt the *ilux* operon
for use in iGEM backbones and for wide use in several bacteria. We chose to present a proof of
concept on the bacteria *Dickeya solani*, a crops devastating bacterium, but more than just
making bacteria luminescent, the FIAT LUX team aims at creating a biosensor tool that is
adaptable to any strain. That way, if a new cultivable phytopathogen bacteria is discovered
tomorrow, we would be able to transform it with our tool, infect a plant with it in a lab,
observe the bacterial propagation thanks to the constitutive bioluminescence, predict its wider
propagation thanks to a mathematical model, and understand it better to react quickly and save
crops. In order to create such a tool, we went through many different steps described on our [Experiments page](https://2022.igem.wiki/insa-lyon1/experiments), as well as
many engineering cycles, described below.

![design](https://static.igem.wiki/teams/4239/wiki/schemas-communs/explication-ilux.png)

**Figure 1** - Diagram explaining the biochemical pathways
generated by the
*ilux* operon. Left: *ilux* operon coding for the proteins below (luciferase, fatty
acid reductase, FMN reductase). Right: enzymatic reaction behind the *ilux* operon

## CREATING BIOBRICKS: SYNTHESIS OF GENES AND *fiatlux* ASSEMBLY

---

### 1. Removal of restriction sites

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

In order to adapt the *ilux* operon for use
in
the iGEM
backbones, **standard iGEM prefixes and suffixes had to be added**; the EcoRI (E) and XbaI (X) restriction sites must be positioned upstream, and SpeI (S) and PstI (P) downstream of the operon. We therefore had
**to remove the EcoRI, XbaI, SpeI, PstI restriction sites from the *ilux* coding
sequence**, so the operon would not be destroyed when using these enzymes. The *ilux* operon
contains 3 PstI restriction sites, 1 EcoRI site and 1 XbaI site that needed to be
removed.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

First, the 5 sites were removed by directed mutagenesis PCR.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

The DNA construction was digested and analyzed by agarose gel electrophoresis. It was unsuccessful: the restriction sites were indeed mutated and the correct fragments were obtained (Figure 2), but due to technical limits, we were not able to reconstruct the whole operon at once.

![](https://static.igem.wiki/teams/4239/wiki/engineering/gel-abe-cd.png)

**Figure 2** - Electrophoresis gel results (0.7% agarose), revealed with EtBr + UV. Lane 1: marker. Lane 2: the fragment at 2.5 kb corresponds to *fiatluxCD*. Lane 3: the fragment at 3.6 kb corresponds to *fiatluxABE*.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

As we were not able to reconstruct the operon, another method to remove the restriction sites needed to be tested.

---

As the problematic restriction sites were not present in the genes *iluxA*, *iluxB* and *iluxE*, we performed a classical PCR to reconstruct *fiatluxABE*. We decided to have mutated *fiatluxCD* synthesized in a pACYDuet-1 vector containing *fiatluxCD* and a constitutive and weak BBa\_J23117 promoter (without restriction sites).

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie2.png)

The amplified sequence of *fiatluxABE* was inserted in pSB1C3 (already in iGEM biobrick format) and pBAD18 (high-copy vector with an arabinose inducible promoter). The synthesis of *fiatluxCD* was ordered.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie2.png)

The DNA constructions were digested and analyzed by
agarose gel electrophoresis. Restrictions profiles were done: the results were in agreement with the expected sequence.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie2.png)

The restriction map shows that *fiatluxABE* was well
reconstructed. The synthetized plasmid containing *fiatluxCD* is correct. In the end, **we obtain *fiatluxABE* in pBAD18 and pSB1C3, and *fiatluxCD* in pACYDuet-1.** The corresponding parts are [BBa\_K4239007](http://parts.igem.org/Part%3ABBa_K4239007) and [BBa\_K4239006](http://parts.igem.org/Part%3ABBa_K4239006).

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie2.png)

---

### 2. Silent mutations effect

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

Silent mutations effect on luminescence production had to
be tested. If light is still emitted, it would mean that the operon is still
functional despite our mutations.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

In order to test the effect of the previously induced
silent mutations, *E.coli* DH5 alpha were transformed with
pACYDuet-1-*fiatluxCD* and pBAD18-*fiatluxABE* (that are compatible).

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

Light emission was observed with ChemiDoc XRS+, Bio-Rad
(a luminescence detection apparatus).

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

After observation with ChemiDoc XRS+, Bio-Rad, it appears
that **we can still observe bioluminescence after having introduced silent
mutations (Figure 3).**

![](https://static.igem.wiki/teams/4239/wiki/engineering/silent-mutation-schema1.png)

**Figure 3** - Luminescent *E.coli* DH5α containing pACYDuet-1-*fiatluxCD*
and pBAD18-*fiatluxABE* captured with a highly-sensitive camera. Left: captured
luminescence. Right: simple photo of the Petri plate

---

### 3. Insertion in a conjugative plasmid

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

Having both *fiatluxABE* and *fiatluxCD* in the
same conjugative vector is essential if we want to be able to transfer it to other
bacterial strains in future experiments. **We had to assemble both parts in the same
conjugative plasmid: pSEVA521**.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

*fiatluxCD* and *fiatluxABE* were then
assembled together in the conjugative vector pSEVA521 by HiFi assembly.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

pSEVA521-*fiatluxCDABE* was amplified, extracted and
verified with a restriction profile.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

The restriction profile was the one expected: **the *fiatlux*
operon is now complete and well inserted in a conjugative plasmid: pSEVA521.**

## DETERMINATION OF THE OPTIMAL STORAGE TEMPERATURE AND ANTIBIOTIC CONCENTRATION OF *E. COLI* TRANSFORMED WITH FIAT LUX

---

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

*fiatlux* was inserted in two conjugative
vectors, pSEVA521 and pSEVA531. They both have different origins of replications:
pSEVA531 is thus present in a higher number of copies. To study the stability of the
plasmids in the long term, **it is necessary to determine the optimal storage
temperature and tetracycline concentration of *E.coli* transformed with *fiatlux***.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

*E.coli* bacteria were transformed by pSEVA521-*fiatlux*
and pSEVA531-*fiatlux* separately and grown in a medium supplemented with 10 µg/mL
of antibiotic. Several temperatures were tested for the storage conditions.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

*E.coli* transformed strains were stored at
different temperatures
(-80°C, 4°C, room temperature) and then revivified on a medium containing an
antibiotic concentrations of 10 µg/m, to see how they recover from storage.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

***E.coli* bacteria did not grow again after
freezing at -80°C.**

---

We chose to test different antibiotic
concentrations to check if it had
an impact on this issue.

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie2.png)

Different antibiotics concentrations were tested, after
storage at
different temperatures.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie2.png)

*E.coli* transformed strains were stored at different
temperatures
(-80°C, 4°C, room temperature) and then revivified on media containing several
antibiotic concentrations (2, 5 and 10 µg/mL).

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie2.png)

*E. coli* strains transformed by
pSEVA531-*fiatlux* showed
slower growth than those transformed by pSEVA521-*fiatlux*, which means **a too
high strength of transcription may exhaust the bacteria. Plus, *E.coli*
transformed with *fiatlux* showed better growth in a 5µg/mL tetracycline
medium, and were able to grow again after any storage temperature at 5µg/mL, but not
at 10µg/mL.**

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie2.png)

## IMPLEMENTATION OF *fiatlux* IN *D.SOLANI*

---

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

After successfully transforming *E.coli*
DH5α bacteria with our
plasmids and characterizing the tool, **the objective was to insert it in
*D.solani***.
The four plasmids used in the experiment are pSEVA521, pSEVA521-*fiatlux*,
pSEVA531 and pSEVA531-*fiatlux*. The two empty vectors (pSEVA521 and pSEVA531)
are used as a control to compare the effects of the *fiatlux* operon in
*D.solani*.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

We first tried to insert the plasmids by electroporation.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

*Dickeya solani* colonies did not grow on a
selective
medium.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

*D.solani* does not respond to electroporation.

---

We used the conjugation method to
transfer the plasmids into *D.solani.*

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie2.png)

An intermediate donor bacteria *E.coli* MFDpir was
transformed,
and then the conjugation was performed.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie2.png)

After 48h of incubation, some colonies grew on the
selective media,
and luminescence was observed with a high sensitivity camera.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie2.png)

**The four plasmids were successfully inserted in *D.solani*,
and the colonies were luminescent when *fiatlux* was present** (Figure 4). The
colonies
were re-isolated on Petri dishes and the most luminescent colonies were selected to
be used for the rest of the experiments.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie2.png)

![](https://static.igem.wiki/teams/4239/wiki/engineering/study-lumi-d-solani.png)

**Figure 4** - Study of the luminescence produced by *D.solani* colonies
containing 4 different plasmids (pSEVA531-*fiatlux*, pSEVA521-*fiatlux*, pSEVA521 and
pSEVA531). The four strains were striked on the same Petri plate and this was repeated four
times, for different tetracycline concentrations (0, 3, 5 and 10 µg/mL). Luminescence can be
observed for all the colonies containing pSEVA531-*fiatlux* and pSEVA521-*fiatlux*.

## MATHEMATICAL MODEL

---

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

To compare the growth between bacteria with or
without plasmid, and
to compare the effect of the different plasmids, we needed to model each situation.
To do so, we used the software R and **several mathematical models to determine the
one that best fitted the experimental growth curves**, get the parameters for
each situation, and compare them thanks to a statistical analysis. The purpose of
the analysis was to **detect if there were significant differences between the
different growth conditions and the different backbones tested.**

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

We modeled the curves to find the appropriate
mathematical model
thanks to the nlstools package on R. Thanks to this package, we could then visualize
them and the model adjusted to them, but also get the parameters of this model. We
modeled both the O.D. 600 nm as a function of time and the Luminescence/OD ratio as
a function of time. Once we did it for all of our conditions and replicates, we
compared them using the emmeans library.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

After extracting data from our experiments, we compared
the AIC
criteria of our different models for each condition (antibiotic concentration and
the nature of the plasmid). We checked that the best model really fitted the curve
properly and returned consistent parameters values. We statistically compared the
parameters of each model. It helped us determine which condition (or backbone) was
significantly different from the other.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

The model that best fitted our curves turned out to be
the Baranyi
model. We observed that pSEVA531-*fiatlux* produced more luminescence than
pSEVA521-*fiatlux* in *Dickeya solani*. Moreover, the *fiatlux* operon
didn’t affect bacterial growth in the pSEVA531 backbone.

## QUANTIFYING THE ENZYMATIC KINETICS OF OUR LUCIFERASE

---

Our team decided to quantify the enzymatic kinetic of our luciferase in order
to better document it. The first step was to purify our luciferase, which is an enzymatic
complex, from bioluminescent bacteria. Then we planned to purify our FMN reductase encoded
by *frp*, and do the reaction catalyzed by the luciferase *in vitro*.

### 1. Extraction of our luciferase

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

The first step was to **extract the proteins
contained in our
bioluminescent bacteria** without denaturing them.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

We chose to extract our luciferase using *E.coli*
MFDpir x pSEVA531-*fiatlux*. We chose *E.coli* MFDpir transformed with
pSEVA531-*fiatlux* and not with pSEVA521-*fiatlux* (lower copy number plasmid
than pSEVA531-*fiatlux*) because we wanted the maximum possible concentration of
our luciferase. We chose to use the “French Press” method for the lysis of our bacteria
because it disrupts cell walls without altering cytoplasmic proteins. The solution
obtained is centrifuged and the proteins remain in the supernatant.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

We verified the presence of proteins in the supernatant
by the
measurement of the absorbance at 280 nm. The absorbance at 280 nm is typical of
the aromatic rings of the amino acids composing proteins.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

**The French Press method works.**

---

### 2. Purification of our luciferase

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

**We wanted to extract our luciferase, in order
to purify it,
from the solution obtained**  after the French Press. To do so, we had to
separate our luciferase from the other proteins. As our protein is not tagged,
the easiest way was to perform a gel exclusion chromatography.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

Our enzymatic complex weighs 80kDa. We used a Sephadex
G-100 gel because it will exclude all proteins above 100kDa and separate proteins under
that size. The size of our column is 20cm tall to have a good separation without using
too much Sephadex gel. We added 1mL of our solution (whose O.D. at 280 nm was 3.301
originally) to be purified. We measured the O.D. at 280 nm of each collected tube to
draw a chromatogram.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

The chromatogram indicated that all our proteins were
eluted in
the first collected tube, which means we overloaded our column.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

We need to add a smaller volume of our solution to be
purified
or we need to dilute our solution.

---

We decided to add a smaller volume of our
solution in a 20cm tall
Sephadex G-100 column.

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie2.png)

We added 350µL of our solution to be purified, instead.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie2.png)

We obtained a satisfactory chromatogram with 6 different
tubes of
2.5mL containing proteins.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie2.png)

**350µL of a solution with an O.D. at 280 nm of 3.301 is
sufficient to not overload a 20cm tall Sephadex G-100 column.**

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie2.png)

---

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie.png)

To know in which one of the 6 tubes the
luciferase is present, we
chose to do a polyacrylamide gel electrophoresis (PAGE).

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie.png)

We did a PAGE non-denaturing with a sample load of 10µL.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie.png)

No bands other than the protein ladder (molecular-weight
size
marker) appeared on the PAGE.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie.png)

**Our collected tubes didn’t contain enough
proteins.**
Indeed, the limit of detection of our gel is 50µg. Hence, we assumed that our
collected tubes had a concentration of protein smaller than 5µg/µL. We needed to
quantify the protein concentration of each tube to know if our hypothesis was
true.

---

To quantify the protein concentration of each
collected tube and
the original solution to be purified, we chose to undergo a Bradford protein
assay.

![design](https://static.igem.wiki/teams/4239/wiki/engineering/design-luxie2.png)

We did the Bradford protein assay.

![build](https://static.igem.wiki/teams/4239/wiki/engineering/build-luxie2.png)

Our collected tubes had protein concentrations that ranged from
0.96µg/µL to 1.07µg/µL while the original solution to be purified had a protein
concentration of 1.20µg/µL.

![test](https://static.igem.wiki/teams/4239/wiki/engineering/test-luxie2.png)

Our hypothesis is true: **our solutions are not concentrated
enough to be detected using a PAGE.** We have to concentrate our solutions.

![learn](https://static.igem.wiki/teams/4239/wiki/engineering/learn-luxie2.png)

  Because our experiment didn’t work, we could not go further in these
experiments. But we imagined and designed a series of experiments that we could have
performed if we had had valid results in the previous experiments. These included for example
changing the promoter in front of the genes *fiatluxA* and *fiatluxB*, with a
high-level expression inducible promoter. In that way we would increase the production of
our luciferase, leading to more luciferase in the solution obtained after the cell lysis by
the French Press. We could use the T7 promoter. More details about the designed experiments
are presented in the [Experiments.
page](https://2022.igem.wiki/insa-lyon1/experiments)

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![banière](https://static.igem.wiki/teams/4239/wiki/schemas-communs/baniere-gauche.png)

# EXPERIMENTS

![banière](https://static.igem.wiki/teams/4239/wiki/schemas-communs/baniere-droite.png)

The accomplishment of our project has been a long process over the course
of almost a year. We have thoroughly followed an experimental strategy to
successfully reach our goals. The experiments we have performed are explained below:

The objective of our project was to create a more universal bacterial tracking system
than the one developed by Gregor et al. (2018). We wanted to:

* make *ilux* compatible with the Biobrick format;
* place *ilux* under the control of a constitutive promoter;
* avoid an energetic cost for the bacterium;
* place the system on a conjugative plasmid with an origin of replication for a wide host
  spectrum.

The *ilux* operon was born from a mutated natural luminescence operon present in
the bacteria *Photorhabdus luminescens*: the *lux* operon, and the addition of the
*frp* gene from the *Vibrio campbellii* bacteria. These mutations were error-prone PCR
induced according to Gregor et al.’s study in 2018, and are listed in the following table. The aim
was to create a system of genes that produces more light.

| *lux* operon genes | Mutations to form *ilux* |
| --- | --- |
| *luxA* | K22E, T119A, S178A |
| *luxB* | S13P, V121A, N259D |
| *luxC* | N10T, N59D, E74D, S256P, M355T, N360D |
| *luxD* |  |
| *luxE* |  |
| *frp* | M213L, R242L, K256R |

*Table 1 : Amino-acids mutations induced to the* lux *operon, by
error-prone PCR, in order to
create the improved version* ilux.

![Schema](https://static.igem.wiki/teams/4239/wiki/schemas-communs/explication-ilux.png)

**Figure 1** From the *ilux* operon to light. Top left: design of the
*ilux* CDABE-frp operon. Bottom left: enzymes encoded by *ilux* . Right panel:
autonomous light emission by luciferase and substrate-forming enzymes. FIAT LUX is based off of
this operon.

The *ilux* operon is composed of 6 genes: *iluxA*, *iluxB*,
*iluxC*,
*iluxD*, *iluxE* and *ilux-frp*, that form an autonomous system producing
bioluminescence
thanks to the encoded luciferase enzymes.
Each one of the *ilux* genes has a specific role, as you can see on the illustration above
(Figure
1). According to Gregor et al.’s study in 2018, an FMN reductase is naturally already present in *E.coli*
bacteria, making the *frp* gene not that essential for a durable luminescence production in *E.coli*.
As for *Dickeya solani*, our proof of concept bacteria, the MAP laboratory has already used the
*lux*
operon without *frp* without any problem, and since *ilux* allows a bioluminescence
production
sevenfold more important than *lux*, we decided not to make it a priority to work with
*frp*
during our experiments. However, we think that adding *frp* to our tool would be an added value
for
future iGEM teams.

## FIAT LUX BIOBRICKS CREATION

## FIAT LUX IN *E.coli*

## FIAT LUX IN *D.solani*

## FIAT LUX *IN SITU*

## TO GO FURTHER

## CREATING A BIOBRICK : FROM *ILUX* TO *FIATLUX*

### 1. Restriction sites removal: Silent mutations induction and reassembly

Initially, the *ilux* operon was available in a [pGEX](http://2022.igem.wiki/insa-lyon1/material) plasmid in an [*E.coli* DH5α](http://2022.igem.wiki/insa-lyon1/material).
The objective of this study was to adapt this *ilux* operon for use in the iGEM
backbones. To assemble BioBricks for iGEM, it is necessary that EcoRI and XbaI restriction
sites are placed upstream, and SpeI and PstI downstream of the operon. The *ilux*
operon contains 3 PstI restriction sites, 1 EcoRI site and 1 XbaI site: these 5 sites need
to be removed (Figure 2).

**We attempted to remove the restriction sites in the *iluxCDABE* genes**
by following these steps: DNA extraction (1), PCR directed mutagenesis (11), agarose gel
analysis with green gel (4), and gel purification (5). The restriction sites were indeed
mutated, so removed, and 6 PCR fragments were obtained, according to Figure 2.A. However, we
were not able to gather the fragments together with an overlap PCR, as the resulting product
was not concentrated enough. We then tried a TEDA cloning to gather all the fragments
together in the [pWSK129](http://2022.igem.wiki/insa-lyon1/material) plasmid (a low copy
plasmid), and also [pGEX(-)](http://2022.igem.wiki/insa-lyon1/material) but the restriction
profiles of the obtained plasmids were both not correct (4).
In another engineering cycle, we tried another method. As the problematic restriction sites
were not present in the genes *iluxA*,
*B*
and
*E*
, we performed a classical PCR to reconstruct
*fiatluxABE* in [pSB1C3](http://2022.igem.wiki/insa-lyon1/material) (already in iGEM
biobrick format) and [pBAD18](http://2022.igem.wiki/insa-lyon1/material) (high-copy vector with
an
arabinose inducible promoter). After *E.coli DH5α* was transformed by both plasmids.
**Therefore, we obtained *fiatluxABE* without promoter in pSB1C3**, and under the
control of the
arabinose inducible promoter pBAD in pBAD18.
The part containing the *iluxCD* genes was synthesized by chemical synthesis
(GeneCust), by removing directly the restrictions sites in the sequence. In
the synthesis, we incorporated upstream *iluxCD* the [BBa\_J23117](http://parts.igem.org/Part%3ABBa_J23117) promoter. It is a
constitutive
and weak promoter, so no inducer needs to be added.

![photo](https://static.igem.wiki/teams/4239/wiki/schemas-communs/pgex-fragment.png)

**Figure 2.A** - *ilux\_pGEX-Amp* plasmid containing *ilux* genes
and EcoRI, PstI,
XbaI restriction sites to be removed. In pink, the PCR primers needed to remove
restriction sites (L1XX). The PCR fragments obtained are numbered from 1 to 6.

![photo](https://static.igem.wiki/teams/4239/wiki/schemas-communs/biobrick-creation.png)

**Figure 2.B** - Scheme representing the major steps to obtain
*fiatluxABE* and
*fiatluxCD*. Step1: directed mutagenesis was performed to remove the 5
restriction
sites present in *iluxCDABE-frp*. Step2: *fiatluxABE* in a biobrick
format. Step3: the
construction was cloned in two different backbones pSB1C3 and pBAD18. Step4:
*fiatlux-CD* was ordered and obtained by chemical synthesis.

### 2. Effect of silent mutations on the luminescence

Since mutations have been introduced in the *ilux* operon, the effect on
bioluminescence had to be tested before reassembling *fiatluxABE* and *fiatluxCD*
in the same plasmid. In order to check the bioluminescence, the two plasmids
pACYDuet-1-*fiatluxCD* and pBAD18-*fiatluxABE* were inserted into *E.coli*
DH5α by
electrotransformation (8). After transformation, the bioluminescence of the transformants
was well observed with [ChemiDoc XRS+,
Bio-Rad](http://2022.igem.wiki/insa-lyon1/material), indicating that the genes *fiatluxABE* and *fiatluxCD* are
expressed and functional (Figure 3).

![photo](https://static.igem.wiki/teams/4239/wiki/experiments/test-lumi-e-coli.png)

**Figure 3** - Luminescent *E.coli* DH5α containing pACYDuet-1-*fiatluxCD*
and pBAD18-*fiatluxABE* captured with a highly-sensitive camera, without
needing to add
arabinose to induce the promoter (basal expression). Left: captured luminescence.
Right:
simple photo of the Petri dish.

Besides, since *fiatluxABE* is under the control of the pBAD promoter,
different arabinose concentrations were added to the culture to determine the link between
inducer quantity and bioluminescence production. The mean of luminescence over the OD at
600nm ratio is shown on Figure 4:

![graph](https://static.igem.wiki/teams/4239/wiki/experiments/graph-lumi-od-arabinose.png)

**Figure 4** - Effect of the inducer concentration on light emission. Mean
value of the luminescence over OD 600 ratio, measured on *E.coli* DH5α
transformed
with pACYDuet-1-*fiatluxCD* and pBAD18-*fiatluxABE*. Arabinose is the
inducer of the pBAD
promoter. Glucose represses the pBAD promoter. Blue plots correspond to
*E.coli*
DH5α transformed with pACYDuet-1-*fiatluxCD* and pBAD18-*fiatluxABE*.
Orange and gray plots
correspond to *E.coli* DHα that possesses only one of the two plasmids. As a
control, only LB wells without bacteria were measured (yellow plots).

This analysis shows that a low concentration of arabinose of 0.02 % is enough
to induce bioluminescence production, but an arabinose concentration of 0.1% inhibits it.
This would imply that high ilux gene transcription could result in an excessive energy cost.
Therefore, **we conclude that it is necessary to use a plasmid with a low-copy origin of
replication in order to avoid depleting the bacterium of the energy needed to operate
the luciferase**. However, in any case, we decided to use a constitutive promoter to
drive lux expression, since we cannot add an inducible substance during an in situ study.

### 3. Assembly of the *fiatluxCD* and *fiatluxABE* parts in the same plasmid

The aim was then to assemble *fiatluxABE* and *fiatluxCD* parts, to
check that the operon was still functional after being mutated and reassembled, and to put
it in an appropriate backbone to be able to transfer our *fiatlux* operon to other
strains, even non-transformable ones, making our tool usable for more potential bacteria.
Besides, we wanted to assemble *J23117-fiatluxCD* and *fiatluxABE* together in a
conjugative plasmid, possessing an origin of transfer. An assembly of the *J23117-fiatluxCD*
and *fiatluxABE* was thus done by HIFI assembly (7) in pSEVA521 and pSEVA531, two
conjugative vectors with RK2 and pBBR origin of replication respectively (low replication
strength). These two plasmids were transformed (8) in *E. coli* DH5α, and cloning was
verified by a restriction profile (6;2;4). The *ilux* assembly of *fiatluxABE* and
*fiatluxCD* was successfully done in both [pSEVA521](http://2022.igem.wiki/insa-lyon1/material) and [pSEVA531](http://2022.igem.wiki/insa-lyon1/material) vectors (Figure 5).

![snapgene](https://static.igem.wiki/teams/4239/wiki/experiments/gibson-pseva.png)

**Figure 5** - Diagram explaining the construction of the FIATLUX plasmid.
Assembly of *J23117-fiatluxCD* and *fiatluxABE* in the pSEVA521 backbone,
obtaining the constructed plasmid pSEVA521-*J23117-fiatluxCDABE* by Hifi. *J23117-fiatluxCD*
fragments were obtained after a PCR on pACYDuet-1-*fiatluxCD* and
*fiatluxABE*
fragments were obtained after a PCR on pBAD18-*fiatluxABE*. Scheme done with
the Snapgene
software.

| *ilux* operon genes | Mutations to form *fiatlux* |
| --- | --- |
| *iluxA* |  |
| *iluxB* |  |
| *iluxC* | A→G(A949A) |
| *iluxD* | A→G(L167L) |
| *iluxE* |  |
| *iluxfrp* | G→C(L60L), A→G(A234A) |
| non coding DNA | 2547\_2548insG, T2689A |

**Table 2:** Directed mutagenesis on the *ilux* operon.
Mutations
in nucleotides in order to create the new Biobrick *fiatlux*. Mutations are
silencious in coding regions. The assembling of *iluxCD* and *iluxABE* by
digestion and ligature into the pSEVA plasmids lead to a guanine insertion between
the nucleotide 2547 and 2548 (from the A of the start codon of *iluxC*). This
insertion is not in a coding region or a ribosome binding site, and thus is not
supposed to change the functionality of the *lux* system.

## PRIMARY CHARACTERIZATIONS OF OUR TOOL *FIATLUX* IN *E.coli*

Our *fiatlux tool* was designed in order to achieve a perfect balance
between luminescence output and regular bacterial growth. It is critical that the presence
of our plasmid does not disrupt the regular functioning of the transformed bacteria;
otherwise, an imaging study using our technology would be inaccurate. A bacterial growth
defect due to our *fiatluxCDABE* system could impact bacterial processes such as
virulence. In order to determine the appropriate culture and storage conditions, it is also
necessary to characterize how the transformed strains react to temperature changes after
cold storage and various antibiotic concentrations.

As an initial stage, optimization and characterisation were carried out in
*E. coli* DH5. We have created 2 plasmids that differ only in their origin of
replication. [pSEVA521-fiatluxCDABE](http://2022.igem.wiki/insa-lyon1/material)
carries out the RK2 replication origin whereas [pSEVA531-fiatluxCDABE](http://2022.igem.wiki/insa-lyon1/material) has the
pBBR1 replication origin. The number of copies of these plasmids were not evaluated in our
studies but based only on previous studies performed in *E. coli* (Jahn et al. 2016)
that evaluated a copy number between 2 and 7 for RK2-based plasmid and between 5 to 10 from
pBBR1 replication origin. The effect on *E. coli* of the *fiatluxCDABE* expression
was evaluated by comparing the phenotypes of *E. coli* strains expressing *fiatluxCDABE*
with those carrying the empty vector pSEVA521 or pSEVA531.

### 1. Effect of tetracycline on the growth and luminescence of *E. coli fiatluxCDABE* strains on solid media.

First, we analyzed the behavior of the *fiatluxCDABE* strains on solid
LB media. We streaked *E. coli* strains on LB agar containing various concentrations of
tetracycline. Because we observed that DH5α with pSEVA531-*fiatluxCDABE* streaked on a
LBagar
plate with tetracycline at 10 µg/ml had difficulties to grow, we decided to determine the
effect of tetracycline on the strains containing pSEVA521-*fiatluxCDABE*,
pSEVA531-fiatluxCDABE or the empty vectors. We also decided to evaluate their growth
depending on storage conditions. Strains were directly streaked from the -80°C glycerol
stock, from colonies stored at 4°C or at room temperature. The plates were then incubated at
37°C overnight. Results are presented in Figure 6.

Transformed *E. coli* DH5α were able to grow after being stored at -80°C
or +4°C, except in the presence of a 10 µg/mL tetracycline medium. When stored at room
temperature, transformed *E. coli* DH5α were able to grow at any tetracycline
concentration. We also observed that DH5α transformed with pSEVA521-fiatlux grew better in a
2 and 5 µg/mL tetracycline LBagar medium. In contrast, DH5α strains containing the empty
vectors pSEVA521 or pSEVA531 grow very well on LBagar with 10 µg/ml tetracycline, even when
the inoculum is from bacteria stored at 4°C or -80°C. Taken together, these observations
indicate that expression of *fiatlux* from pSEVA531 certainly delays the ability of the
*E.coli* DH5α to resist an usual concentration of tetracycline since the use of lower
concentrations of tetracycline improves growth. It is likely that the production of
light by luciferase has an energy cost that would prevent metabolically inactive
bacteria (as stored at 4°C or -80°C) from adapting to the presence of tetracycline.

![snapgene](https://static.igem.wiki/teams/4239/wiki/results/temperature-and-antibiotics.png)

**Figure 6.A** - A: *E.coli* transformed with
pSEVA531-J23117-*fiatluxCDABE*, B: *E.coli* transformed with
pSEVA531-*J23117-fiatluxCDABE*, C: *E.coli* transformed with an empty
pSEVA531, D:
*E. Coli* WT.
Antibiotics and temperature characterizations of *E.coli* strains transformed
with
*fiatlux* (A and B). For each tetracycline concentration (2, 5 or 10 µg/mL), a
different storage temperature was tested (-80°C, 4°C or RT), and the effect on
luminescence was observed.

![snapgene](https://static.igem.wiki/teams/4239/wiki/results/lumi-temperatures-and-antibiotics.png)

**Figure 6.B** - Same petri dishes as Figure 6.A but with luminescence.
Pictures taken with NightSHADE LB 985 In Vivo Plant Imaging System

**From now on, a tetracycline concentration between 2 and 5 µg/ml should
be used for E.coli DH5α transformed strains culture**.

### 2. Effect of tetracycline on the growth and luminescence of *E.coli fiatluxCDABE* strains on liquid media.

We also compared the growth over time (O.D.) of *E. coli* DH5a
containing pSEVA521, pSEVA531, pSEVA521-fiatluxCDABE and pSEVA531-fiatluxCDABE, with a TECAN
microplate reader. Luminescence was also measured over time. Bacteria were cultivated in LB
at 37°C with shaking microplates. To note that we concentrated our [bio modelisation](http://2022.igem.wiki/insa-lyon1/material) analysis on
*Dickeya solani* behavior and not on *E.coli*’s. Thus, all of our *E.coli*
graphics and results are not statistically confirmed but based on observation, owing to time
constraints.
![snapgene](https://static.igem.wiki/teams/4239/wiki/results/do-521.png)

**Figure 7** - O.D. of *E.coli* transformed strains with pSEVA521
vectors in function of time. Blue data correspond to pSEVA521-fiatlux transformed
strains, red data correspond to empty pSEVA521 transformed strains. All data have
different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles
to
10µg/mL. In this graphic, only one replicate is shown among the 16 performed
replicates,
for each condition. The represented replicate is representative of the global
results.

![snapgene](https://static.igem.wiki/teams/4239/wiki/results/do-531.png)

**Figure 8** - O.D. of *E.coli* transformed strains with pSEVA531
vectors in function of time. Blue data correspond to pSEVA531-fiatlux transformed
strains, red data correspond to empty pSEVA531 transformed strains. All data have
different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles
to
10µg/mL. In this graphic, only one replicate is shown among the 16 performed
replicates,
for each condition. The represented replicate is representative of the global
results.

In this graphic, only one replicate is shown among the 16 performed
replicates, for each condition. The represented replicate is representative of the global
results.

Concerning pSEVA521 transformed strains, *fiatlux* seems to slightly
affect the maximum O.D., but a statistical analysis would be necessary to confirm its
significance.
Concerning pSEVA531 transformed strains, pSEVA531-fiatlux transformed bacteria and empty
pSEVA531 transformed bacteria in Tet 0µg/mL seems to have similar maximum O.D., whereas a
growth lag seems to occur in Tet 10µg/mL transformed bacteria. Indeed, the beginning of
growth is visible starting from 7 000 secs for empty pSEVA531 transformed bacteria, and from
75 000 secs for pSEVA531-fiatlux transformed bacteria. Therefore, it seems that pSEVA521 is
a better vector to use in *E.coli*.

Then, the luminescence over growth ratio is analyzed to understand how *fiatlux*
transformed *E.coli* bacteria behave, in function of the used vector (pSEVA531 or 521)
and in function of the antibiotic concentration (tet 10, 5 or 0 µg/mL).

![snapgene](https://static.igem.wiki/teams/4239/wiki/results/lumin-do-521.png)

**Figure 9** - Luminescence over O.D. ratio of *E.coli* transformed
strains with pSEVA521 vectors in function of time. Blue data correspond to
pSEVA521-fiatlux transformed strains, red data correspond to empty pSEVA521
transformed
strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to
5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among
the
16 performed replicates, for each condition. The represented replicate is
representative
of the global results.

![snapgene](https://static.igem.wiki/teams/4239/wiki/results/lumin-do-531.png)

**Figure 10** - Luminescence over O.D. ratio of *E.coli* transformed
strains with pSEVA531 vectors in function of time. Blue data correspond to
pSEVA531-*fiatlux* transformed strains, red data correspond to empty pSEVA531
transformed
strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to
5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among
the
16 performed replicates, for each condition. The represented replicate is
representative
of the global results.

These graphics show that, whatever the tetracycline concentration is, *E.coli*
DH5alpha transformed with *fiatlux* emits luminescence, and *E.coli* bacteria that
do not have our *fiatlux* operon do not produce any light. It is interesting to note
that, in a tet 0µg/mL growth medium, there is no selective pressure on the transformed
bacterias anymore, but these bacterias were still shown to produce luminescence. Besides,
for all antibiotics concentration, pSEVA521-*fiatlux* transformed bacteria’s behavior
seems
similar, whereas pSEVA531-*fiatlux* transformed bacteria’s behavior is irregular, hence
not
reproducible.

To note that the luminescence is comparable between pSEVA521-*fiatlux*
transformed bacteria and pSEVA531-*fiatlux* transformed bacteria. Besides, by comparing
both
O.D and luminescence/O.D. ratio graphics, the maximum luminescence emission is observed at
the end of the exponential growth phase, which confirms that luminescence is potentially
dependent on the internal bacterial metabolism.

In the end, both liquid and solid media showed that the plasmid pSEVA521
seemed more suitable than pSEVA531 in *E.coli* DH5α strains. We believe that the
reason could be the following: a higher replication rate could tire the bacteria and
thus slow down its growth, which could be due to the potential toxic effect the high
production of luminescence could have on the bacteria.

## PROOF OF CONCEPT IN A PHYTOPATHOGENIC BACTERIA : *D.solani*

After transforming *E. coli* bacteria with our *fiatlux* operon, we
transferred the plasmids into phytopathogenic bacteria *Dickeya solani*, to
validate our proof of concept.
After having characterized the stability of this new strain, we infected plants (chicory
and Saintpaulia) in order to visualize the luminescence of the bacteria directly *in
situ*, thus validating our proof of concept.

### 1. Inserting the plasmids into *D.solani*

![experiments8](https://static.igem.wiki/teams/4239/wiki/experiments/experiments8.png "Figure 8: Scheme of the steps of the transformation of our plasmids (pSEVA521, pSEVA521-fiatlux, pSEVA531 and pSEVA531-fiatlux) into E.coli MFDpir and conjugation into D.solani. ")

**Figure 11**: Scheme of the steps of the transformation of our
plasmids (pSEVA521, pSEVA521-fiatlux, pSEVA531 and pSEVA531-fiatlux) into
*E.coli*
MFDpir and conjugation into *D.solani*.

After successfully transforming *E.coli* DH5α bacteria with our plasmids and
characterizing the tool, our next goal was to insert it in *D. solani*.
We used the WT strain *D. solani* D s0432-1.
The four plasmids used in the experiment are pSEVA521, pSEVA521-*fiatlux*, pSEVA531
and pSEVA531-*fiatlux*.
The two empty vectors (pSEVA521 and pSEVA531) act as control and are used to compare the
effects of the *fiatlux* operon in *D.solani*.
We performed two methods to introduce our tool in *D.solani* D s0432-1: an
electrotransformation, and a conjugation.

For the electrotransformation, we used Petri dishes with 5µg/mL of tetracycline to only
select bacteria with the plasmid.
As a result, we got a few colonies that grew on Tetracycline 5µg/mL, those transformed
with pSEVA 531, and those transformed with pSEVA531-*fiatlux*.
However, none of the colonies transformed with pSEVA531-*fiatlux* were luminescent.
Although our results were not conclusives, we did not push our investigation further,
because our second method of DNA transfer worked.

For the conjugation, we used an intermediate *E. coli* strain, MFDpir, because of
several advantages it presented for the conjugation:

* The growth of the bacteria requires DAP (Diaminopimelic acid), which is mixed with
  LB. *E.coli* MFDpir can grow only in the presence of DAP because the strain is
  dapA-, a mutation causing DAP auxotrophy.
* Its genome encodes a RP4 conjugation machinery, which makes conjugative pili
  required for conjugation with other bacteria.This machinery recognises the oriT of
  the pSEVA plasmids.

We thus used a transformation (8) to insert our four plasmids in *E.coli* MFDpir.
After 48h of incubation at 37°C, some colonies grew on the Petri dishes (with 5µg/mL of
tetracycline) and the luminescence of colonies was observed with a highly-sensitive
camera.

At this point, it was possible to carry out the conjugation step (14), between
*E.coli* MFDpir and *D.solani*, for the four plasmids.
After 48h of incubation at 30°C, some colonies grew on the Petri dishes (with 5µg/mL of
tetracycline) and the luminescence of the colonies was observed with a ChemiDoc XRS+,
Bio-Rad highly-sensitive camera.
For each conjugation, 7 colonies were re-isolated on solid LB.

In order to make sure the colonies we obtained after conjugation were *D.solani*,
we performed a pectinase test (25).
We spread a drop of our 7 clones (putting in liquid culture the day before) on a Petri
dish containing 0.2% of dipecta, and after 24h, we revealed with copper acetate.
According to the stains around the drops, all of our colonies are *D.solani*.
**The four plasmids were successfully transfered into *D.solani*, and the
colonies were luminescent.**

![experiments20](https://static.igem.wiki/teams/4239/wiki/experiments/experiments20.png "Figure 20: Test Pectinase with D.solani D. s0432-1 carrying out plasmid pSEVA521, pSEVA521-fiatlux, pSEVA531 or pSEVA531-fiatlux, and the WT strain D s0432-1 without plasmid.")

**Figure 12:** Test Pectinase with *D.solani* D. s0432-1
carrying out plasmid pSEVA521, pSEVA521-fiatlux, pSEVA531 or pSEVA531-fiatlux,
and the WT strain D s0432-1 without plasmid."

### 2. Characterization of *fiatlux* in *D.solani*

**2.1. Growth and Luminescence of *D. solani fiatluxCDABE* Strains on Solid Media**

Growth and luminescence of the *Dickeya solani* strains in function of tetracycline
concentration in LB agar medium were evaluated as it was done previously with *E.
coli*.
The strains were stored at a temperature of -80°C, and plated on Petri dishes.
Four conditions were tested, for each strain: 0, 3, 5 and 10µg/mL of tetracycline.
The Petri dishes were incubated at 30°C.
Photos of the Petri dishes were taken after 24h and 48h of incubation with a ChemiDoc
XRS+, Bio-Rad highly-sensitive camera, to see the growth of colonies.
In order to compare the behavior of our tool in *D.solani* with our results for *E.
coli*, we did the same test in parallel with both *D.solani* and *E. coli*
DH5α strains.

![experiments26](https://static.igem.wiki/teams/4239/wiki/experiments/experiments26.png)

![experiments27](https://static.igem.wiki/teams/4239/wiki/experiments/experiments27.png "Figure 26:<i>D.solani</i> strains containing the four plasmids (pSEVA521, pSEVA521-<i>fiatlux</i>, pSEVA531 and pSEVA531-<i>fiatlux</i> isolated on Petri Dishes (with 0, 3, 5 and 10 µg/mL of tetracycline) after 24h and 48h (top: exposure time 0.1 sec; daylight - bottom: exposure time 60 sec; without light)")

**Figure 13** - *D.solani* strains containing the four
plasmids (pSEVA521, pSEVA521-*fiatlux*, pSEVA531 and
pSEVA531-*fiatlux* isolated on Petri dishes (with 0, 3, 5 and 10 µg/mL of
tetracycline) after 24h and 48h (top: exposure time 60 sec; daylight - bottom:
exposure time 60 sec; without light).

![experiments28](https://static.igem.wiki/teams/4239/wiki/experiments/experiments28.png)

![experiments29](https://static.igem.wiki/teams/4239/wiki/experiments/experiments29.png "Figure 28:<i>E. coli</i> DH5alpha strains containing the four plasmids (pSEVA521, pSEVA521-<i>fiatlux</i>, pSEVA531 and pSEVA531-<i>fiatlux</i> isolated on Petri Dishes (with 0, 3, 5 and 10 µg/mL of tetracycline) after 24h and 48h (top: exposure time 0.1 sec; daylight - bottom: exposure time 60 sec; without light)")

**Figure 14** - *E. coli* DH5alpha strains containing the four
plasmids (pSEVA521, pSEVA521-*fiatlux*, pSEVA531 and
pSEVA531-*fiatlux* isolated on Petri Dishes (with 0, 3, 5 and 10 µg/mL of
tetracycline) after 24h and 48h (top: exposure time 60 sec; daylight - bottom:
exposure time 60 sec; without light).

* **Effect of our *fiatlux* operon**

The bacteria containing the plasmid with *fiatlux* (pSEVA521-*fiatlux* and
pSEVA531-*fiatlux*) and the bacteria with the empty vectors (pSEVA521 and pSEVA531)
look similar (same shape and same size), for a same concentration of tetracycline.
This means that the expression of  **our *fiatlux* operon**  from *J23117*
promoter in pSEVA521 and pSEVA531  **does not affect the growth of *Dickeya
solani*** .
This is a big difference from what was observed in *E. coli* DH5α.

* **Effects of Tetracycline Concentration on Luminescence and Growth**

**Luminescence of *D.solani* (with pSEVA521-*fiatlux* and pSEVA531-*fiatlux*)
is much stronger with a higher concentration of tetracycline.**
This is an unexpected result since *fiatlux* is under the control of a constitutive
promoter.
We suggest two hypothesis to explain this result:

* 1- This may be due to higher transcription of the *fiatlux* operon due to
  transcriptional readthrough from the promoter of the tetA resistance gene present on the
  plasmids pSEVA521 and pSEVA531.
  The more tetracycline is added to the medium, the more the resistance gene could be
  expressed.
  Since the tetA gene and the *fiatlux* operon are oriented in the same direction, a
  change in the transcription of the first gene could modify the transcription of the
  second, even if a transcriptional terminator is placed between the two.
* 2- Another hypothesis is a change in the copy number of the plasmid as a function of
  the
  antibiotic concentration.
  This has been observed in the past with other plasmids (Chong et al. 2003).

However, we have not conducted experiments to understand the causes of this change in
luminescence as a function of tetracycline concentration.
It would be interesting to do so in the future.
The antibiotic concentration does not affect the shape and size of the isolated
colonies.
However, we can notice a difference in the speed of growth.
By observing pictures of our strains after 24h, we notice that we can already observe
isolated colonies on LB medium, but not on medium containing tetracycline.
Tetracycline 10µL/mL is the medium where the bacteria grew the least.
After 48h, we can see isolated colonies on each media.

* **Effect of the Plasmid: Comparison between pSEVA521-fiatlux and
  pSEVA531-fiatlux**

As for *E.coli* DH5⍺, **D.solani with the plasmid pSEVA531-*fiatlux*
emits more luminescence than the strain with pSEVA521-*fiatlux*.**
pSEVA531-*fiatlux* being a higher copy number plasmid than pSEVA521-*fiatlux*,
the luciferase is more produced and more light is emitted.

We did a second experiment in order to characterize our tool: the growth and
luminescence of colonies on solid medium were observed every 30 min for 48h thanks to a
NightShade LB 985 (Berthold) optical imaging system, equipped with a high resolution CCD
camera.
The experiment with *D.solani* and *E.coli* DH5alpha containing the plasmids
(pSEVA521, pSEVA521-*fiatlux*, pSEVA531 and pSEVA531-*fiatlux*) on Petri
dishes in LB medium with different concentrations of tetracycline (0, 5 and 10 µg/mL)

Video of *D.solani* and *E.coli* DH5alpha containing the
plasmids (pSEVA521, pSEVA521-*fiatlux*, pSEVA531 and pSEVA531-*fiatlux*) on Petri
dishes in
LB medium with different concentrations of tetracycline (0, 5 and 10 µg/mL)

What we can observe in the time laps: *Dickeya* transformed with
pSEVA531-*fiatlux*
with LB media and tet 5µg/mL media start to emit luminescence before all the others.

* **Effects of Tetracycline Concentration on Luminescence**

We can clearly see the effect on antibiotic concentration on the time laps: for both LB
medium and tetracycline 5µg/mL medium, luminescence appears earlier than with
tetracycline 10 µg/mL medium.
This may be due to the growth delay we observed for higher antibiotic concentrations.
Luminescence reaches a peak, before fading.
It is fading faster on LB medium than on medium with antibiotics.
This can be explained by the selective pressure of tetracycline that encourages the
bacteria to keep the plasmid.
In order to quantify the speed of the loss of the plasmid, we will perform a stability
test on *D.solani* with our four plasmids.

* **Comparison between *E.coli* DH5⍺ and *D.solani***

Luminescence evolves over time: for both *D.solani* and *E. coli* DH5alpha,
the luminescence starts to increase until it reaches a maximum, before decreasing.
For a same concentration of tetracycline and the same plasmids, *D.solani* emits
the maximum of luminescence earlier than *E. coli* DH5⍺. These maxima of
luminescence also reach higher levels.

* **Comparison between Isolated Colonies and Bacterial Mat**

This video allows us to observe the dynamics of bacterial growth on solid medium and the
luminescence of colonies over time.
We observed that the luminescence is emitted longer for isolated colonies than for the
bacterial mat.
After 48h, the bacterial mats are almost no longer luminescent.
It can be assumed that the luminescence is affected by the growth state of the bacterium
which varies according to the cell density on the solid culture medium.
Bacteria in the bacterial mat are stuck to each other.
They have to share the nutrients of the medium and the oxygen.
While isolated bacteria have a greater availability of nutrients and oxygen.

Based on the results, we could conclude that  **pSEVA531-*fiatlux* is more
efficient than pSEVA521-*fiatlux* with *D.solani* because it generates more
light without affecting bacterial growth.**

**2.2. Effect of tetracycline on the growth and luminescence of *D. solani
fiatluxCDABE*
strains on liquid media**

We then performed the characterization of luminescent strains of *Dickeya
solani* in liquid culture in the same way as it was done with *E. coli*.
The objectives were to:

* identify and determine the ideal parameters, so as to maximize the
  luminescence, without disturbing the bacterial growth.
* compare the bacterial growth and the luminescence of the bacteria with
  plasmids containing *fiatlux* to the bacteria with the empty vector, to
  determine the impact of *fiatlux*.
* compare the effect of antibiotic concentration on the growth of bacteria
  and on the luminescence

In order to reach these goals, we followed the OD (at 600 nm) and the
luminescence of the bacterial cultures for 48 hours, thanks to a microplate
reader.
Bacteria containing each of our four plasmids are put in a liquid culture with
different concentrations of tetracycline (0, 3, 5, 7 and 10 µg/mL).
All the cultures were added at the same OD, and we did 4 to 8 replicates for
each condition, so we can perform statistical analysis.
Thanks to these data, we performed a mathematical modelisation of the bacterial
growth, in order to compare the different conditions

The results of this experiment can be found on the page  **Results : [Model](http://2022.igem.wiki/insa-lyon1/model).**

### 3. Stability Test of the Plasmids in *D.solani*

To test the stability of our plasmid in *D.solani*, we performed a
stability test (20) for each plasmid.
In the absence of selection pressure (the antibiotic), the plasmid could be lost
during cell divisions.
This control is essential for iGEM teams wishing to ensure that the plasmid
remains stable in their bacteria.

The objective is to determine if after a certain number of generations without
selective pressure, enough bacteria still possess the plasmid.
A generation of bacteria is defined as the division of one bacteria into two
bacteria.
At every generation, the number of bacteria is multiplied by two. For example,
in a chicory leaf, after beginning of infection, between 10 and 15 generations
are sufficient to infect all the leaf.
In our experiment in vitro in LB liquid culture, we set up the experiment to
obtain 9 cell divisions of *D. solani* per day.
This can be calculated by measuring the OD of the culture at 600 nm at the
beginning of the culture (DO(0) = 0,0012) and after 24 of incubation (DO(f) =
0,600).
We performed the test over three days, to obtain a total of 27 generations.
Every day, we enumerated the bacteria cells of the liquid culture that grew on
two different media (LB and tet), but we also monitored the evolution of
luminescence (produced by pSEVA531-*fiatlux* and pSEVA521-*fiatlux*).
Bacteria growing in LB medium represent the total number of bacteria in the
liquid culture and bacteria growing in LB with 5µg/mL of tetracycline represent
only bacteria of the culture still containing the plasmid.

This experiment was realized with two replicates that are consistent. In order
to do statistics, a third replicate has to be realized (not done here because of
a lack of time).

The results of this experiment can be found on the page  **Results : [Wetlab](http://2022.igem.wiki/insa-lyon1/wetlab)**.

## TRACKING BACTERIA : *IN SITU* OBSERVATION

After characterizing *D.solani* with our four plasmids, we performed infections of
chicory leaves and potatoes (23).
The objectives are multiples:

* To verify whether or not symptoms appear while infection with *D.solani*
  containing our plasmids.
* To monitor plant infection by following the luminescence thanks to the software and
  the hardware designed by our team.
* To understand if plasmids containing the *fiatlux* operon (pSEVA521-fiatlux
  and pSEVA531-*fiatlux*) have effects on plant infections by *D.solani*,
  compared to the empty vectors (pSEVA521 and pSEVA531).

### 1. Chicory infections

Firstly, we infected chicory (23), because this plant can be easily and rapidly
infected, and its small size enables us to make a lot of replicats.
*Dickeya solani* carrying pSEVA521, pSEVA521-*fiatlux*, pSEVA531 or
pSEVA531-*fiatlux* were used to infect chicory leaves, with eight replicates for
each.
Four chicories were used: their leaves were mixed to have homogeneous leaf sizes between
the replicates and to avoid a disturbance of results due to more resistant chicory.
The size of the infection sites were measured after 30 and 48h, and the luminescence of
the leaves was observed with a ChemiDoc XRS+, Bio-rad highly-sensitive camera.

![experiments9](https://static.igem.wiki/teams/4239/wiki/experiments/experiment9.png "Figure 9 : Average sizes of infection stain of chicory by D.Solani DS0432-1, with different plasmids (8 replicates per plasmid)")

**Figure 15:** Average sizes of infection stain of chicory by
*D.Solani* DS0432-1, with different plasmids (7 replicates per plasmid)

![experiments21](https://static.igem.wiki/teams/4239/wiki/experiments/experiments21.png "Figure 21: Graph of the size of infection strain of chicories by D.Solani DS0432-1, with different plasmids (7 replicates per plasmid), 30 and 48 hours after infection.")

**Figure 16:** Graph of the size of infection strain of
chicories by *D.Solani* DS0432-1, with different plasmids (7 replicates per
plasmid), 30 and 48 hours after infection.

*D. solani* with plasmids pSEVA531 or pSEVA531-*fiatlux* showed more important
signs of infection than *D.solani* with pSEVA521 and pSEVA521-*fiatlux*.
Indeed the average size of infection is significantly higher for pSEVA531 and
pSEVA531-*fiatlux* (after both 30h and 48h).

Moreover, we can observe a significant difference of the size of the rotten area between
*D.solani* with pSEVA521 and pSEVA521-*fiatlux*.
On the other hand, there is no significant difference between the size of the rotten
area between *D.solani* with pSEVA531 and pSEVA531-*fiatlux*.
The behavior of the infection by *D.solani* is impacted by *fiatlux* when used
in pSEVA521, but not when used in pSEVA531. All the test has been performed with the
software R using R studio.
In order to calculate the pvalue, we used the multiple tukey test, enabling us to
compare each single pair of means.
For further details about the code, please refer to: [Model](https://static.igem.wiki/teams/4239/wiki/experiments/stats-endives.pdf)
[Data](https://static.igem.wiki/teams/4239/wiki/experiments/taille-endives.pdf)

Moreover, infection with bacteria containing pSEVA531-*fiatlux* produces much more
luminescence than with bacteria containing pSEVA521-*fiatlux*.
**This observation confirmed that for *D.solani*, the plasmid
pSEVA531-*fiatlux*
is more efficient than pSEVA521-*fiatlux* (regarding symptoms of infection and
luminescence).**

Timelapse of *D. solani* containing pSEVA531-*fiatlux* or pSEVA531 done with
the NightSHADE LB 985 In Vivo Plant Imaging System was realized, by taking pictures
every 30 minutes to follow the evolution of both the luminescence and the symptoms.

On this video, we can observe the evolution of symptoms over time: the rot is growing
all over the leaf, and reaches the edge of the leaf for one of them. As far as
luminescence is concerned, we observe a front of luminescence where the bacteria are
degrading the tissues. We can suppose there are more bacteria at this front, or more
nutrients available, so more energy for the bacteria, and therefore more luminescence.
After some time, luminescence is fading in the most rotten parts of the leaf. All those
observations are complemented by a further analysis thanks to the software designed by
our team.

### 2. Potato Infections

Potato tuber were infected, with only *D.solani* containing plasmid pSEVA531
and pSEVA531-*fiatlux*. Five potatoes were infected for each plasmid. After 24h
and 48h after the infection, half of potatoes were cut to observe the putrefaction
in them, and the luminescence, thanks to the ChemiDoc XRS+, Bio-Rad and the
NightSHADE LB 985 In Vivo Plant Imaging System.

![experiments19](https://static.igem.wiki/teams/4239/wiki/experiments/experiments19.png "Figure 19 : Potato tuber infections with D.solani containing the plasmids pSEVA531 or pSEVA531-fiatlux, observed with the NightShade (left : exposure time : 0,1 sec; dayligth - right : exposure time 60 sec; without light)")

**Figure 17:** Potato infections with *D.solani*
containing the plasmids pSEVA531 or pSEVA531-*fiatlux*, observed with the
NightSHADE LB 985 In Vivo Plant Imaging System (left : exposure time : 0,1
sec; dayligth - right : exposure time 60 sec; without light)

As far as symptoms are concerned, we can observe a rotten area of approximately the
same size for each tuber. For one of the control potatoes, infected with the empty
vector, the symptoms are more severe. Other replicates will be performed in order to
study those symptoms. Luminescence is well observed for potatoes infected with
bacteria containing *fiatlux* operon, and not with empty vectors, as desired.

### 3. Conclusion

We succeeded in infecting chicories and potatoes with our plasmids. We could observe
luminescence only when *D.solani*, used for infections, owns the *fiatlux*
operon on its plasmid.
The next step would be to infect potato plants or another living plant, to observe
how bacteria spread themself into tissues and the different parts of the plants. It
would be also interesting to infect a potato tuber and let it grow, to observe the
bacteria during the whole life cycle of the plant.

## LUCIFERASE CHARACTERIZATION, OTHER PLASMIDS, OTHER BACTERIA...

### 1. Conjugaison of our plasmid in other bacteria

*fiatlux* is made as an analyzing tool, to help scientists to better understand the
behavior of new or unwell known pathogens.
We value the adaptativity of our tool, and in order to offer our tool to researchers, we
want to make sure it can adapt to as many bacteria as possible.
This is why we inserted our plasmid pSEVA531-*fiatlux* in other bacterium genus.
We chose pathogens with different assets, in order to open opportunities for other
studies.
Here are the ones we tried so far:

* ***Citrobacter rodentium***: a laboratory mice pathogen.
  Bioluminescence imaging (BLI) has already been used to determine the *in vivo*
  colonization dynamics of *C. rodentium*.
  We decided to focus on this bacteria in order to enable future comparisons between
  our tool and other bioluminescent imaging solutions that exist.
* ***Pseudomonas putida***: it can be encountered in diverse
  ecological habitats.
  It has a remarkably versatile metabolism, adapted to withstand physicochemical
  stress, and the capacity to thrive in harsh environments.
  Owing to these characteristics, there is a growing interest in this microbe for
  industrial use, therefore, it seemed interessant for us to focus on this pathogen.
* ***Vibrio natriegens***: has the fastest growth rate of any known
  bacterium.
  This property makes it interesting to open up other kinds of studies with our tool:
  understanding the properties of the bacteria *in vivo*.
* ***Pseudomonas protegens***: it is a model organism used in
  plant-microbe interactions, biological control of phytopathogens.

![new experiments14](https://static.igem.wiki/teams/4239/wiki/experiments/new-experiments14.png "Figure 14 : results of the conjugation of pSEVA531 and pSEVA531-fiatlux in Citrobacter rodentium DBS100 (A & B) and in Pseudomonas putida KT2440 (C & D), on Petri Dishes with 10µg/mL of Tetracycyclin (left (A & C): exposure time 60 sec; without light - right (B & D): exposure time : 0,1 sec; dayligth). The disposition of plasmids is the same on all pictures.")

**Figure 18 -** Results of the conjugation of pSEVA531 and
pSEVA531-fiatlux in *Citrobacter rodentium* DBS100 (A & B) and in *Pseudomonas
putida* KT2440 (C & D), on Petri dishes with 10µg/mL of Tetracycycline
(left (A & C): 0,1 sec; dayligth - right (B & D): exposure
time : exposure time 60 sec; without light). The disposition of plasmids is the same
on all
pictures.

We decided to insert our plasmid with the conjugation method, because it is easy to
implement and has a great chance of success.
Furthermore, we already had the bacteria *E.coli* MFDpir with our four different
plasmids (discussed in the preceding section), which sped up the experiment.

The conjugation gave us colonies for all of the four bacterial strains we tested.
Then, the bacteria were observed with the ChemiDoc XRS+ Bio-rad highly-sensitive camera.
Luminescence was observed only for two strains: *Citrobacter rodentium* and *Pseudomonas
putida*.
For those two strains, luminescence was oberserved for bacteria containing plasmid
pSEVA531-fiatlux, and not for those containing the empty vector.
This means that our *fiatlux* operon is well responsible for the luminescence
observed.

These results show that our tool can be inserted in other bacteria than *D.solani*
(in this case *Citrobacter rodentium* and *Pseudomonas putida*).
However, the characterisation and the stability of pSEVA531-*fiatlux* have to be
determined in future experiments.

### 2. Creation of new biobricks

After having a proof of concept by infecting chicory with *D.solani*
containing our plasmids, we wanted to create new biobricks, in order to improve our
tool.

**2.1. Offer a Biobrick without Promoter**

![experiments10](https://static.igem.wiki/teams/4239/wiki/experiments/experiments10.png "Figure 10 : construction of biobrick fiatluxCDABE without the promoter J23117")

**Figure 19:** Construction of biobrick *fiatluxCDABE*
without the promoter *J23117*

The objective was to create a biobrick with *fiatlux*, that we could offer to other
igem team or researcher, by enabling the use of our tool with any promoter.
For example, they can add promotor with a higher copy number, or an inducible promoter
(unlike the promoter used in the project).

A digestion (2) enables us to remove the promoter J23117 and an annealing of two primers
(20) was made to ligate both plasmid ends.
The new vector has a unique BamHI restriction usptream *iluxC* that will be useful
for further cloning of new promoters.

![experiments22](https://static.igem.wiki/teams/4239/wiki/experiments/experiments22.png "Figure 22 : Upstream sequence of fiatlux without promoter in pSEVA531")

**Figure 20:** Upstream sequence of fiatlux without promoter in
pSEVA531

**2.2. Toxin/antitoxin system**

![experiments11](https://static.igem.wiki/teams/4239/wiki/experiments/experiments11.png "Figure 11 : construction of pSEVA531-fiatlux-Axe/Txe  and pSEVA531-fiatlux-Hok/Sok")

**Figure 21:** Construction of pSEVA531-fiatlux-Axe/Txe and
pSEVA531-fiatlux-Hok/Sok

We then wanted to couple the *fiatlux* operon with a toxin/antitoxin system in the
same plasmid.
This enables a constitutive selection of the plasmid to be carried out: this method of
selection ensures that all the bacteria present on the plant have the plasmid, and can
hence emit light.
Toxins have a longer life span in cells than antitoxins, so if the bacteria lose their
plasmid, the toxin is no longer destroyed by the antitoxin (Van Melderen 2010).
This results in the death of the bacteria, thus assuring the death of all *fiatlux*
non-carriers.
As a consequence, by involving a selective pressure, the toxin/antitoxin system
increases the stability of the plasmid in the bacteria.

We tried to clone 2 differents toxin/antitoxin systems : hok/sok and axe/txe.
Both systems should be inserted in pSEVA531-fiatlux, which was digested (2), before the
fiatlux operon.
axe/txe was inserted in the opened plasmid thanks to a TEDA cloning (9).
hok/sok, contained in pGEX, was transfered in the plasmid pSB1C3 thanks to a TEDA
cloning (9). Then hok/sok was inserted in pSEVA531 thanks to a ligation (3).
*E. coli* DH5α were transformed by both plasmids.

After verification thanks to a PCR on colonies (10), we can conclude that only the
plasmid pSEVA53-*fiatlux*-Axe/Txe worked, but not the pSEVA53-*fiatlux*-Hok/Sok.
In order to make sure that our transformation with pSEVA53-*fiatlux*-Axe/Txe
worked, we also did a restriction map, which showed coherent results.
We also plan to sequence our plasmid.

**2.3. Creation of a Plasmid by replacing *fiatlux* by *lux***

![experiments12](https://static.igem.wiki/teams/4239/wiki/experiments/experiments12.png "Figure 12 : construction of pSEVA531-<i>lux</i>")

**Figure 22:** construction of pSEVA531-*lux*

The objective of this plasmid was to replace the *fiatlux* gene by the *lux*
gene (Gregor et al. 2018), to compare the difference in emitted luminescence between *fiatlux*
and *lux*.
Indeed, although we could show the efficiency of our *fiatlux* system to make
bacteria luminescent, we could not compare if *fiatlux* was more luminescent than
the *luxCDABE* system currently commonly used in laboratories.
This plasmid construction did not work.
We leave room for future reworkings and improvements of our project for future iGEM
teams.

**2.4. Insertion of *fiatlux* in a Plasmid with a Kanamycin Resistance Gene
(did not work: to be developed further by future iGEM teams…)**

![experiments13](https://static.igem.wiki/teams/4239/wiki/experiments/experiments13.png "Figure 13 : construction of pSEVA221-<i>fiatlux</i>")

**Figure 23:** Construction of pSEVA221-*fiatlux*

The objective of this plasmid is to see the effect of the new antibiotic resistance gene
on the bacterial growth and on the luminescence.
Indeed, we observed during our project that the level of luminescence varies according
to the concentration of tetracycline used in the medium to maintain a selecting
pressure.
Changing the type of resistance gene would allow us to determine if the observed
phenomenon is only related to tetracycline resistance.
Kanamycin is a bacteriostatic antibiotic, which means that it kills bacteria rather than
inhibiting their growth as a bacteriostatic antibiotic such as tetracycline does.
Unfortunately, this plasmid construction did not work.
We leave room for future reworkings and improvements of our project for future iGEM
teams.

### 3. Enzymology: luciferase characterization

The aim of this part was to purify the luciferase encoded by *fiatluxAB* and the
FMN-reductase encoded by *frp*, and to characterize the enzymatic kinetics of the
luciferase encoded by *fiatluxAB*.

**3.1. Extraction of our luciferase**

Our luciferase was extracted from *E.coli* MFDpir containing the plasmid
pSEVA531-*fiatlux*. *E.coli* MFDpir transformed with pSEVA531 (high copy
number plasmid) was chosen and not with pSEVA521 (low copy number type) because we
wanted the highest possible concentration of our luciferase.
The lysis of the bacteria (16) was done using the “French Press” method.
This method was chosen because it disrupts cell walls without altering cytoplasmic
proteins.
The solution obtained is centrifuged and the proteins remain in the supernatant.
The supernatant is collected: this is our solution A.

**3.2. Purification of our Luciferase**

* **Gel exclusion chromatography**

Our solution A contains all the proteins from our *E.coli* MFDpir x
pSEVA531-*fiatlux*.
Our luciferase encoded by *fiatluxAB* has to be separated from the other proteins.
It is an enzymatic complex that weighs 80kDa with two subunits of ~40kDa (Meighen et
al., 1996). Because we want to use our luciferase after the purification, we have to
purify it without denaturing it.

A Sephadex G-100 column was used to realize a gel exclusion chromatography (17). The
exclusion size of the Sephadex G-100 column is 100kDa. In that way, all proteins heavier
than 100kDa will be excluded from the column and eluted first.

A column of 20cm tall was built, dissolving the gel in a solution of distilled water
containing 0.03% of azide sodium (17). The O.D. 280 nm of the solution A is of 3.301.
This measure is necessary to have a global idea of the quantity of proteins contained in
it.

The column is loaded with 1mL of the solution A (17). The fraction collector is set to
collect 2.5mL of eluent in each tube.

The O.D. 280 nm is measured for each collected tube in order to draw the chromatogram.
The chromatogram showed a single peak meaning that the column was overloaded. The
separation of the proteins was unsuccessful.

A new 20cm tall Sephadex G-100 column was built as previously. 350µL of the solution A
is loaded. 30mL of the eluent were collected in 12 tubes of 2.5mL. The chromatogram
obtained is shown below.

![experiments23](https://static.igem.wiki/teams/4239/wiki/experiments/experiments23.png "Figure 23 : Chromatogram obtained with a 350µL solution A loaded on a 20cm tall Sephadex G-100 column")

**Figure 24:** Chromatogram obtained with a 350µL solution A
loaded on a 20cm tall Sephadex G-100 column

The O.D. 280nm of a solution of 350µL of the solution A added to 2.5mL of the
eluent is 0.394. The first tube collected contains the majority of the proteins (O.D. 280nm
= 0.214). After the 6th tube, the O.D. 280nm drops to almost 0, meaning that all proteins
have been eluted. The first six tubes were conserved at -20°C.

* **Polyacrylamide gel electrophoresis (PAGE)**

A sample of each of the 6 tubes collected after the Sephadex G-100 column
must be used in a polyacrylamide gel electrophoresis (PAGE) (18) in order to know the size
of proteins contained in each tube.

Because the luciferase is a complex composed of two subunits, the gel must be
non-denaturing. 10µL of each tube were deposited in the gel with a loading buffer (18).

No bands other than the protein ladder (molecular-weight size marker)
appeared. Either our tubes had no proteins or the protein concentration was too weak. The
detection threshold is at 50µg of proteins deposed. Hence, the protein concentration of each
tube was assayed using the Bradford method (19).

* **Protein assay - Bradford method**

A standard range of BSA was created (19) (Fig.X). The R² is 0.9953 which is
satisfactory. The equation is “O.D.(595nm)=0.0193\*concentration” with the concentration in
µg/mL.

![experimentsBradford](https://static.igem.wiki/teams/4239/wiki/experiments/experimentsbradford.png "Figure 25 : Chromatogram obtained with a 350µL solution A loaded on a 20cm tall Sephadex G-100 column")

**Figure 25:** Standard range of BSA for the Bradford protein
assay.

The protein concentration of two samples (n=2) of each tube are dosed using the Bradford
method (19). The O.D. 595 nm is measured and used to calculate the protein concentration
using the equation of the standard range of BSA. In order to have the real concentration
in each tube, the factor 250 must be applied to the concentration calculated because a
dilution of 250 was done on each sample in the protocol (19).

![experiments24](https://static.igem.wiki/teams/4239/wiki/experiments/experiments24.png "Figure 24 : Protein concentration in each tubes (n=2)")

**Figure 26:** Protein concentration in each tubes (n=2)

The protein concentration of each collected tube and of the solution A range between
0.96 and 1.20 µg/µL. Because 10µL was deposed in the gel, the amount of protein in the
gel ranged from 9.6 to 12.0 µg, which is under the threshold value of 50µg. Hence,
proteins had to be concentrated in each tube. Because our solvent is aqueous, we thought
of using Amicon ® Ultra centrifugal filters on our collected tubes to concentrate our
solutions. However, even if this will allow us to see proteins on a PAGE, the amount of
proteins collected in each tube will be too low for further use.

**3.3. Conclusion**

We didn’t have the time to continue our experiments. If we had more time, we would have
changed our plan and switched the promoter in front of *fiatluxA* and
*fiatluxB* for a high-level expression inducible promoter. This and the following
plan for the enzymatic characterisation is presented in below.
However, **this experiment shows that the amount of luciferase present in our
bioluminescent bacteria is too low for direct purification.**

### TO GO FURTHER …

In order to purify and verify the presence of our luciferase in the collected fractions
in a more optimal way, we thought about a new series of experiments that we could have
performed if we had enough time. The first thing to do would be to place the gene
encoding for the luciferase under the control of a strong inducible promoter, such as
the T7 promotor, in order to overexpress the protein. Then we would have performed a
PAGE to check this overexpression. This way, we would obtain highly concentrated
fractions after the column purification.

The next step would be to produce an in vitro reaction that would allow us to
characterize the luciferase’s kinetics by measuring the disappearance of only one
reagent (n-decanal), as it satisfies the required conditions to measure the activity of
our protein of interest. To follow the disappearance of the n-decanal, we planned to do
a spectrometric monitoring by choosing the wavelengh at which the n-decanal has the best
absorbance.

Then, we would draw the evolution of the OD for different initial concentrations of
n-decanal throughout time. From these graphs we would acquire information about the
reaction speed (represented by the curve’s slope). Then, if we assemble all these datas
we could plot a Michaelis-Menten hyperbole (reaction speed depending on initial
n-decanal concentration). This representation would allow us to deduce different
constants (Km, Vmax) that characterize our luciferase.

![experiments25](https://static.igem.wiki/teams/4239/wiki/experiments/experiments25.png "Figure 26 : Design for the analysis of the in vitro experiments")

**Figure 27:** Design for the analysis of the *in vitro*
experiments

Here are all the protocols that we have followed to successfully conduct the experiments of our project!

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