Recipes for Solutions and Media

2x YT-medium
16 g Bacto tryptone
10 g Bacto yeast extract
5 g NaCl
were dissolved in 1 L dH₂O, the pH was adjusted to 7.0.
For solid medium 12 g of Bacto agar (per Liter) was added.

50x TAE Buffer stock solution
242 g 2M Tris base
51,1 mL glacial acetic acid (17,4M)
200 mL 100mM EDTA (pH 8)
were dissolved in 1L dH₂O.

Loading buffer for gel electrophoresis
A 0.2M EDTA and 0.05% (w/v) orange G or bromphenoleblue solution was prepared in 50% glycerine.

Ampicillin stock solution
10 g ampicillin sodium salt was dissolved in 100 mL dH₂O, sterile filtered, aliquoted and stored at -20 °C.

Chloramphenicol stock solution
3.4 g chloramphenicol was dissolved in 100 mL Ethanol, sterile filtered, aliquoted and stored at -20 °C.

Clavulanic acid
1 g clavulanic acid was dissolved in 100 mL dH₂O, sterile filtered aliqoted and stored at -20 °C.

Tetracyclin stock solution
5 g Tetracyclin was dissolved in 100 mL Ethanol, sterile filtered aliqoted and stored at -20 °C.

TFB 1, pH 5.7 for preparation of competent cells
0.3 g calciumchloride, 0.6 g potassiumacetate, 1.2 g rubidiumchloride, 1 g manganesechloride x 4 H₂O and 15 mL glycerin were dissolved in dH₂O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.

TFB 2, pH 8.0 for preparation of competent cells
2.2 g calciumchloride, 0.12 g rubidiumchloride, 0.21 g morphoslinopropanesulfuricacid and 15 mL glycerin were dissolved in dH₂O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.

N-(butyryl)-homoserine lactone stock solution
1.7 mg N-butyryl-homoserine lactone was dissolved in 1 mL dimethyl sulfoxide. The solution was sterile filtered.

N-(3-oxododecanoyl)-homoserine lactone
2.8 mg N-3-oxododecanoyl-homoserine lactonce was dissolved in 933 µL dimethyl sulfoxide. The solution was filter sterilized.

List of used Strains

List of used Primers

E. Coli Culture Conditions

For the cultivation of E.coli 2xYT medium was used. If necessary 1µL/mL of an antibiotic stock solution was added. The cultures were incubated at 37 °C and in case of liquid cultures shaken at 250 rpm.

E. Coli Continuous Cultivation

Continuous cultivations were conducted in a 2 L stirred tank bioreactor system (Applikon) with one six-bladed disc turbine impeller. Bioreactors were inoculated with mixed cultures which were derived from mixing different ratios of overnight monocultures to an OD₅₂₀ of 0.5. Growth temperature (37±0.1°C), aeration rate (2.5 L min⁻¹), agitation speed (350 min^-1) and pH value (pH 7.0) were automatically kept constant. The working volume was 1 L and the dilution rate was adjusted to 0.5 h^-1.
Depending on the mode of growth (regulated or unregulated) the medium was supplemented with ampicillin or chloramphenicol, respectively. Samples were taken every hour to monitor the cell density. To determine the ratios of the two strains in the culture, samples were spread out on agar plates, incubated at 37 °C and obtained colored colonies were counted.

Measurement of Growth Curves

Overnight cultures in 20 mL 2xYT medium containing chloramphenicol were inoculated directly from -80°C glycerol cell stock. Cells were grown in non-baffled-flasks at 37°C and 250 rpm.
The next day, main cultures in 75 mL 2xYT medium containing ampicillin were inoculated from overnight cultures to an OD520=0.5. Growth of each strain was observed with and without induction of the promoters regulation the ampicillin resistance gene. All cultures and measurements were conducted as duplicates.
For the induction of beta-lactamase (ampR) expression by P_Rhl und P_Las autoinducers N-buturyl-homoserine lactone and N-3-oxododecanoyl homoserine lactone were added respectively to a final concentration of 10 µmol/L. Cells were grown in non-baffled flasks at 37°C and 250 rpm.
Samples from each flask were taken at appropriate time points depending on the growth phase of the cells. Optical density was measured at a wavelength of 520 nm in order to avoid/minimize absorption by the chromoproteins (see absorption spectra). Cells were left to grow until the stationary phase was reached.

Preparation of competent cells

Chemically competent cells
100 mL of 2xYT Media containing required antibiotic were inoculated and incubate at 37°C and 250 rpm until OD₆₀₀ = 0.5. Subsequently the cells were incubated on ice for 15 minutes and then centrifuged at 4 °C and 400 rpm for 5 minutes. The excess was discarded and the pellets resuspended in 7.5 mL ice cold TFB1. Afterwards the solution is incubated on ice for 90 minutes and then centrifuged for 5 minutes at 4000 rpm. Finally the pellets were resuspended, aliquoted and quick-frozen in liquid nitrogen. The vials were stored at -80°C.

Electrocompetent cells
A liquid culture of E. Coli was grown to an approximate OD₅₂₀ of 0.5 and subsequently incubated on ice for 30 min. Afterwards the cells were spun down for 15 min at 4000xg and 4 °C. The cell pellet was washed with 200 mL icecold sterile MiliQ water and resuspended. Centrifugation and resuspension was repeated followed by another centrifugation at 4000xg and 4°C for 20 min and subsequent resuspension in 50 mL 10 % (v/v) icecold glycerol. The cells were then spun down one last time for 10 min and 3220xg and resuspended in 400 µL icecold 10 % (v/v) glycerol. The now electrocompetent cells were aliquoted, quick-frozen in liquid nitrogen and stored at -80°C.

Cryopreservation

The respective strain was grown on 2xYT medium at 37 °C and 250 rpm overnight. Afterwards a 20 % Glycerin solution was prepared with the liquid culture, aliquoted, shock frozen and stored at -80 °C.

Minipreparation of Plasmid DNA

Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.
First 2 mL of the cell suspension were spun down for 10 minutes (5000g).
For the alkaline lysis of the DNA the pellet was resuspended in 250 μL solution I (RNAseA). 250 μL of solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 g for 10 minutes.
Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000g). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10.000g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000g) and dried.
At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000g).

PCR

Colony PCR
After successful transformation single colonies were picked and analyzed by colony PCR. Therefor each colony was picked from the original plate with a pipette tip and dipped into the PCR-mix. Afterwards the pipette tip was used to inoculate a second agarplate. PCR fragments were analyzed via gelectrophoresis.

DNA amplification via PCR with NEB Phusion® High-Fidelity DNA Polymerase

Gel Electrophoresis

Depending on the size of the DNA fragments a 0.8 to 2% agarose gel with 0.2 µg/mL ethidiumbromide was used. The gel was placed in an electrophoresis chamber and covered in 1x TAE buffer. Subsequently DNA samples mixed with loading buffer were loaded to the gel pockets and separated at 80-120 V for about 2 h. Last the gel was photographed for documentation.

Gel Slice Preparation

10 µL of 6x loading buffer were added to digested DNA und mixed. 55 µL of the sample were loaded onto a 1% agarose gel and separated via gel electrophoresis at 75 mA for 20-40 min depending on the size of the DNA. For gel extraction the gel was visualized on an UV-lightsource and the appropriate bands were cut using the x-tracta™ Gel Extractor tool (Promega). Gel purification was carried out using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.

Cloning

Restriction digest
For restriction of vector and insert DNA approximately 1000 ng purified DNA, 5 µL 10x buffer and 1 µL of each of the two restriction enzymes were mixed in a 1.5 mL reaction tube. The amount of DNA used depended on the size of the desired Fragment. Water was added to a final volume of 50 µL. Restriction was carried out at 37 °C for 1 h. The reaction was stopped by inactivating the enzymes at 80 °C for 20 min.
The buffer was chosen depending on the combination of restriction enzymes used:
- PstI/EcoRI HF: NE 2.1
- PstI/SpeI HF: NE 2.1
- PstI/XbaI: NE 3.1
- all other combinations: NEB CutSmart Buffer

Dephosphorylation
For the desphosphorylation of vector DNA 5.56 µL Antarctic Phos Reaction Buffer (NEB) and 0.5µL Antarctic Phosphatase (NEB) were added to the digestet DNA and mixed. DNA was incubated at 37 °C for 60 min. After 30 min of incubation 0.5 µL Antarctic Phosphatase was added to the DNA and mixed.

DNA Purification
After desphosphorylation the vector DNA was purified using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.

Ligation
For ligation of the constructed plasmid 50 ng vector DNA, 3 times as much insert DNA, 2 µL T4 Ligase Buffer (NEB), 0.5 µL T4 Ligase (NEB) and water added to a final volume of 20 µL were mixed in 1.5 mL reaction tube. Ligation was carried out at 16 °C over night. The reaction was stopped by inactivating the enzymes at 65 °C for 20 min.

Transformation of chemocompetent cells via heatshock
One -80°C glycerol stock of chemocompetend cells was thawed on ice. 10 µL of the ligated DNA was prepared in a 1.5 mL reaction tube. 50 µL of the cells were added and incubated for 20 min on ice. The mixture was then heat shocked at 42 °C for 1 min and cooled on ice for 2 min. 1 mL SOC-medium was added and the cells were incubated for 1 h at 37 °C while shaken at 600 rpm. 100 µL of the cell suspension were plated on a 2xYT agar plate with the according antibiotic (chloramphenicol or ampicillin) and incubated at 37 °C over night.

Electroporation

One -80°C glycerol stock of chemocompetend cells was thawed on ice. 100 pg of purified DNA were prepared in an 1.5 mL reaction tube. 50 µL of the cells were added to the DNA and gently mixed. The cells were transferred into a precooled electroporation cuvette. Electroporation was carried out at 1.8 kV for 5.6 ms. The cells were resuspended in 1 mL S.O.C.-medium, transferred into a 2 mL reaction tube. The cells were incubated at 37 °C and shaken at 600 rpm for 1 h.
100 µL of the cell suspension were plated on a 2xYT agar plate with antibiotic (chloramphenicol or ampicillin) and incubated at 37 °C overnight.

Extraction of Chromoproteins

50 mL of overnight culture were pelleted for 10 min at 3220 g. The supernatant was discarded and the cells were resuspended in 4 mL of 1x phosphate buffered saline (PBS).
Desintegration of the cells was performed by sonication (HD2200 Sonopuls, MS72 sonotrode, 50% performance, 2 x 2 min). The cell suspension was kept on ice during sonication and chilled for 5 min between the sonication cycles. The cell debris were pelleted for 5 min at 16,100 g. The supernatant containing the soluble chromoproteins was sterile filtered (0.2 µm) and stored at 4 °C.

Measurement of Absorption and Emission Spectra of the Chromoproteins

Absorption Spectra
In order to measure the absorption spectra of the different chromoproteins 100 mL 2xYT medium containing chloramphenicol was inoculated with E. coli XL1 Blue MRF' including pSB1C3 with chromoprotein expression cassette and grown over night at 37°C and 250 rpm in non-baffled flask to limit oxygen transfer in order to enhance chromoprotein expression. The whole culture volume was centrifuged for 10 min at 6000 rpm and the supernatant discarded. Cells were resolved in 5 mL PBS and disrupted using ultrasonic technology (see protein purification). Subsequently the cells were centrifuged again at 6000 rpm the supernatant was transferred into a new tube and measured with the nanodrop in a range between 190-840 nm.

Emmision Spectra
For the measurement of the emission wavelength the Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) was used.

Imaging of Cells producing Chromoproteins

Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM N-buturyl homoserine lactone (P_Rhl) or N-3-oxododecanoyl homoserine lactone (P_Las) at 37°C and 250 rpm over night. The medium was inoculated directly from -80°C glycerol cell stock. Cells were diluted with fresh 2xYT medium containing ampicillin and suspension was placed between to agar layers containing ampicllin as well.
Cell growth and division were observed for 3 hours by exciting chromoproteins every five minutes at specific wavelength (see absorption spectra) using YFP und mCherry filters.

Detection of Autoinducers

For the production of autoinducers liquid cultures (2xYT medium containing Chloramphenicol) of E. Coli JM109 bearing the BioBrick BBa_K1073034 and E. Coli TOP10F’ bearing the construct BBa_K1073035 were inoculated and incubated overnight. Once the producing cultures reached an OD520 of about 11, cells were harvested by centrifugation for 10 min at 4000 rpm. The biomass was discarded and the supernatant sterilized by filtration. Subsequently the supernatant was diluted 1:2 with sterile 2x YT medium containing ampicillin.
For the detection of the autoinducers cultures of the reporter strains E. coli JM109 pSB406 for N-(butyryl)-homoserine lactone detection and E. coli JM109 pSB1075 for N-(3-oxododecanoyl)-homoserine lactone detection were grown overnight on 2x YT medium supplemented with Ampicillin. The next day the cultures were diluted 1:15 with the above described supernatant solution and incubated for 3 h at 37 °C and 300 rpm in a 96 well plate.
For calibration of the system a serial dilution of autoinducer stock solution was incubated with the reporter strains as described above.
The bioluminescence, which was developed by the reporter strains depending on the autoinducer concentration was measured with a microtiterplate reader.

Agar Diffusion Test

Overnight cultures of E. Coli JM109 carrying BBa_K1073034 and E. Coli TOP10F’ carrying BBa_K1073035 were grown on 2xYT medium containing ampicillin and the respective autoinducer. The next day the cells were harvested by centrifugation at 4000 rpm for 10 min and subsequently resuspended in fresh 2xYT medium. The obtained cultures were then spread out on ampicillin containing agar plates. A filter paper impregnated with the respective synthetic autoinducer solution was placed on top of the freshly spread agar plate. Afterwards the plates were incubated at 37 °C for 3-4 h and pictures were taken to document the circles of bacterial growth around the filter.
This experiment was also conducted with the supernatant of the respectively other strain as the two strains cross induce each other.

References:
Winson, M. K., S. Swift, L. Fish, J. P. Throup, F. Jørgensen, S. R. Chhabra, B. W. Bycroft, P. Williams, and G. S. A. B. Stewart. 1998. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol. Lett. 163:185–192.