Long culture requirements for Labrys portucalensis :

3–4 days on TSA.

5–6 days in TSB to reach OD > 1.

Preparation (D-5 and D-1)

  • D–5: Started L. portucalensis in 250 mL TSB at 28 °C.
  • D–1:
    • Inoculated 100 mL TSB + Gm (10 µg/mL) with strains EGE 453 (pSEVA621), EGE 460 (pSEVA631), EGE 681 (pSEVA651), and EGE 682 (pSEVA661). Incubation: 37 °C.
    • Inoculated 250 mL TSB + Kan (50 µg/mL) with strain EGE 214. Incubation: 37 °C.

Conjugation Day

Procedure

  • Measured OD of all cultures.
  • Mixed 20 OD units from each culture (volume = 20/OD), max. 50 mL per Falcon.

Mixtures prepared:

  1. L. portucalensis + EGE 214 + EGE 453
  2. L. portucalensis + EGE 214 + EGE 460
  3. L. portucalensis + EGE 214 + EGE 681
  4. L. portucalensis + EGE 214 + EGE 682
  5. L. portucalensis + EGE 214
  6. EGE 453 + EGE 460 + EGE 681 + EGE 682

Pellet prep:

  • Centrifuged 10 min @ 8000 rpm.
  • Removed all supernatant (pipette to eliminate droplets).
  • Washed once with 20 mL TSB, centrifuged again 10 min @ 8000 rpm.
  • Resuspended in 100 µL TSB + 20 µL TSB → very small volume.

Filter mating:

  • Used sterile 0.45 µm filters (blue supports removed with flamed forceps).
  • Deposited on TSA plate, spread cell mixture on top.
  • Let dry near flame/under PSM.
  • Incubated overnight at 30 °C.

Day +1 (Post-Conjugation)

  • Recovered filters into 100 µL TSB in Eppendorfs, vortexed.
  • Plated 100 µL of each conjugation mix on TSA + Gm (30 µg/mL) + Amp (100 µg/mL). Spread with sterile beads.
  • Controls: 5 µL of each donor culture plated directly on TSA + Gm30 + Amp100.

Results after 5 days at 30 °C

  • L. portucalensis + EGE 214 → 5 colonies.
  • L. portucalensis + EGE 214 + EGE 453 → many colonies (+++).
  • L. portucalensis + EGE 214 + EGE 460 → lawn growth.
  • L. portucalensis + EGE 214 + EGE 681 → many colonies (+++).
  • L. portucalensis + EGE 214 + EGE 682 → no growth after 10 days.
  • EGE 453 + 460 + 681 + 682 → many colonies (+++).
  • Controls → negative.

Re-isolation and Further Analysis

Re-isolation: 4 colonies from each conjugation on TSA + Amp100 + Gm30, 30 °C.

Results after 1 week (Re-isolations)

  • L. portucalensis + EGE 214 → no colonies.
  • L. portucalensis + EGE 214 + EGE 453 → 2 colony morphotypes.
  • L. portucalensis + EGE 214 + EGE 460 → 2 colony morphotypes.
  • L. portucalensis + EGE 214 + EGE 681 → 2 colony morphotypes.
  • EGE 460 + EGE 453 + EGE 681 + EGE 682 → no growth.

Two types observed:

  • Small colonies → likely contaminants.
  • Large mucoid colonies → re-isolated on TSA + Amp100 + Gm30 → growth after 1 week.

Plasmid Analysis

Liquid Cultures

  • Started 10 mL TSB with L. portucalensis (control).
  • Started 10 mL TSB + Amp100 + Gm30 with colonies from conjugations (L. portucalensis + EGE 214 + EGE 460 and L. portucalensis + EGE 214 + EGE 453). Incubated at 30 °C.

Day +6: Minipreps

  • Performed minipreps (Promega kit). Note: pellet dissolved in supernatant → re-centrifuged.
  • Elution: 100 µL TE, incubated 30 min at 70 °C.

Nanodrop Results

  • L. portucalensis → 67.8 ng/µL.
  • L. portucalensis + EGE 214 + EGE 453 → 18.8 ng/µL.
  • L. portucalensis + EGE 214 + EGE 460 → 113.3 ng/µL.

Gel Electrophoresis (1% agarose, 30 min @ 100 V)

  • L. portucalensis: no band (expected).
  • L. portucalensis + EGE 214 + EGE 453: faint band.
  • L. portucalensis + EGE 214 + EGE 460: strong band.

Transformation and Verification

Transformation

  • DH5α transformed with 5 µL minipreps (from 453 and 460 conjugations).
  • Plated dilutions (10⁰, 10⁻¹, pellet) on GL + Gm7.
  • Negative control: competent cells without plasmid.

Results

  • L. portucalensis + EGE 214 + EGE 460 → 4 colonies (10⁰), ~30 colonies (pellet).
  • L. portucalensis + EGE 214 + EGE 453 → no colonies.

Plasmid Extraction from Positive Transformants

  • Picked 2 colonies from DH5α transformants (L. portucalensis + EGE 214 + EGE 460).
  • Cultured 2 × 5 mL 2YT + Gm7 overnight at 37 °C.
  • Miniprep (Promega kit), eluted 2 × 50 µL in AE (Macherey-Nagel).

Nanodrop

56.4 ng/µL.

Restriction Digests

Reaction mix:

  • 21.5 µL plasmid DNA
  • 0.5 µL EcoRI
  • 0.5 µL EcoRV
  • 2.5 µL custom buffer

Samples:

  1. Plasmid extraction from L. portucalensis.
  2. Plasmid extraction from L. portucalensis + EGE 214 + EGE 460/DH5α.
  3. Digested plasmid from conjugation (EcoRI + EcoRV).
  4. Digested plasmid from L. portucalensis (EcoRI + EcoRV).
  5. DNA ladder.

Gel (1% agarose, 30 min @ 100 V)

  • Conjugation plasmid (460/DH5α, digested) → 2 bands (1 kb + 2 kb) = expected result.
  • L. portucalensis (control) → no bands (expected).
  • Conjugation plasmid (undigested) → 2 bands, consistent with expected size.
Agarose gel electrophoresis
Figure 1: Agarose gel electrophoresis of plasmid extractions from L. portucalensis conjugants after EcoRI/EcoRV digestion.
Plasmid map
Figure 2: Map of the plasmid used for L. portucalensis transformation: 950 nt between EcoRI and EcoRV.

Time course DH5α transformed with the biosensor construct

We finally obtained the fully assembled biosensor construct cloned into a low copy plasmid (pSEVA261)after ordering it (albeit quite late), which allowed us to directly transform E. coli DH5α cells.

Biosensor construct
Figure 3: The biosensor construct ordered and directly used for DH5α transformation.

E. coli DH5α Transformation

  • Thaw competent Heat Shock E. coli DH5α on ice.
  • Add 3 µL plasmid suspension, incubate 10 min on ice.
  • Heat shock: 42 °C, 1 min, then 10 min on ice.
  • Recovery: 1 mL LB, 30–60 min, allows expression of resistance cassette.

Plating

  • Spin: 4,000 g, 2.5 min.
  • Remove supernatant, leave ~100 µL, resuspend pellet.
  • Plate on kanamycin agar: spread ~150 µL on half the plate with a spreader, then use same spreader for other half.
  • Incubate overnight at 37 °C.

The transformed bacteria were then grown in precultures to perform a miniprep to assess that the plasmid is actually in the bacteria.

Miniprep

  • Follow NEB Miniprep kit protocol (cf: Experiment: Plasmid miniprep).
  • Final elution: 30 µL.
  • Plasmid purified by miniprep was sent to Microsynth for sequencing.

Nanodrop Measurement

33.8 ng/µL (1 µL used for measurement). Total DNA obtained: ~980 ng.

Sequencing Alignment

Sequence alignment
Figure 4: Alignment of the miniprep sequenced with the original sequence of the biosensor construct.

The alignment is perfect, except for a single mutation in a non-coding region (visible as a small discontinuity in the blue line over the construct).

Time-Course Measurement

The preculture of transformed bacteria was diluted 1:100 into a 96-well plate containing various inducer concentrations, in order to determine the conditions for optimal promoter activation and to assess the robustness of the construct. After induction, the plate was incubated for ~1 h before initiating the time-course measurement with the TECAN reader.

96 well layout
Figure 5: 96 well layout for Time course.
Tecan device
Figure 6: Tecan device.

Measurements were taken every 20 minutes, including OD at 600 nm, GFP fluorescence (excitation 485 nm, emission 535 nm, automatic gain), mCherry fluorescence (excitation 570 nm, emission 645 nm, automatic gain), and luminescence (integration time = 250 ms).

Unfortunately, the Tecan did not function properly throughout the time-course experiment, leaving us with data covering only 1 hour and 30 minutes.

The condition at 50 µM IPTG and 50 ng/mL of anydrotetracycline was the best tradeoff between strong signal and not toxicity (good growth curve).

The condition with 20 µM IPTG and 10 ng/mL anhydrotetracycline represented the best trade-off between a strong signal and low toxicity (as indicated by a good growth curve).

Case of double induction

Normalized mCherry signal
Figure 7: Normalized mCherry signal in DH5α transformed with either the biosensor construct or the empty plasmid, under double induction (20 µM IPTG + 10 ng/mL aTc ) or without induction.
Normalized GFP signal
Figure 8: Normalized GFP signal in DH5α transformed with either the biosensor construct or the empty plasmid, under double induction (20 µM IPTG + 10 ng/mL aTc ) or without induction.
Normalized Luminescence signal
Figure 9: Normalized Luminescence signal in DH5α transformed with either the biosensor construct or the empty plasmid, under double induction (20 µM IPTG + 10 ng/mL) or without induction.

* : p < 0,05 between the condition “induction” and “non induction”, ** : p < 0,01 for the same conditions. # : p < 0,05 between the condition “induction” and “Empty plasmid”, ## : p < 0,01 for the same conditions. † : p < 0,05 between the condition “empty plasmid” and “non induction”, †† : p < 0,01 for the same conditions.

It can be seen that, despite the leakiness of the pLac and PTet promoters, which drive reporter fluorescence significantly above the control level, luminescence remains very low and does not differ significantly from the control. A marked increase in luminescence is observed only upon simultaneous addition of both inducers.

Case of single induction (IPTG or aTc)

Single induction IPTG

Normalized GFP signal with IPTG
Figure 10: Normalized GFP signal in DH5α transformed with either the biosensor construct or the empty plasmid, under single induction (20 µM IPTG) or without induction.
Normalized luminescence signal with IPTG
Figure 11: Normalized luminescence signal in DH5α transformed with either the biosensor construct or the empty plasmid, under single induction (20 µM IPTG) or without induction.

It can be seen that the pLac promoter exhibits clear leakiness. Despite this, a stronger GFP signal is observed under inducer conditions.

Regarding the luminescence signal, although it is higher than the control condition initially, it tends to approach the control level as E. coli growth progresses.

Single induction aTc

Normalized mCherry signal with aTc
Figure 12: Normalized mCherry signal in DH5α transformed with either the biosensor construct or the empty plasmid, under single induction (10 ng/mL aTc) or without induction.
Normalized luminescence signal with aTc
Figure 13: Normalized luminescence signal in DH5α transformed with either the biosensor construct or the empty plasmid, under single induction (10 ng/mL aTc) or without induction.

Regarding the PTet promoter, a slight leakiness can be observed; however, the mCherry signal is substantially higher than that measured in the uninduced condition (which reflects a little promoter leakiness). However, a very strong luminescence expression is observed, which is unexpected because in our construct, luminescence should not occur if only one promoter is active. This can be explained by the significant leakiness of the pLac promoter, such that when PTet is induced with aTc, both promoters are effectively active, resulting in a strong luminescent signal.

Comparison of luminescence output

Comparison of luminescence output
Figure 14. Normalized luminescence signal in E. coli DH5α transformed with either the biosensor construct or the empty plasmid, under different induction conditions: single induction with 10 ng/mL aTc or 20 µM IPTG, double induction with 20 µM IPTG and 10 ng/mL aTc, or without induction.

* : p < 0,05 between the condition “Induction 20µM IPTG + 10 ng/mL aTc” and “non induction”, ** : p < 0,01 for the same conditions. # : p < 0,05 between the condition “Induction 20µM IPTG + 10 ng/mL aTc” and “Induction 10ng/mL aTc”, ## : p < 0,01 for the same conditions. † : p < 0,05 between the conditions “Induction 10ng/mL aTc” and “empty plasmid”, †† : p < 0,01 for the same conditions.

Dual induction remains the most effective way to achieve a luminescent signal, which is higher than that observed under single induction with either IPTG or aTc. Although single induction with aTc produces a strong luminescent signal due to pLac leakiness, it is still significantly lower than the signal observed under dual induction. These results validate our construct while highlighting the need for further optimization.

Response at t = 18h

Luminescence at 18h
A
mCherry at 18h
B
GFP at 18h
C
Figure 15 : Normalized luminescence (A) signal, normalized mCherry signal (B) and normalized GFP signal (C), in E. coli DH5α transformed with either the biosensor construct or the empty plasmid, under different induction conditions: single induction with 10 ng/mL aTc or 20 µM IPTG, double induction with 20 µM IPTG and 10 ng/mL aTc, or without induction (NI), at t = 18h.

A final measurement was taken at t = 18 h. Overall, the observed values were in line with expectations. Interestingly, a stronger mCherry response is observed when both promoters are active, whereas GFP shows the opposite trend. Regarding the luminescent signal, which is the main output of this biosensor, a strong signal is detected in the presence of both inducers, as well as in the presence of aTc alone due to pLac leakiness. Furthermore, a notable GFP signal is observed in the absence of inducer, well above the empty plasmid control, which is not the case for mCherry, confirming that pLac exhibits significant leakiness.