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Protocols

In this section you will find detailed protocols of experimental procedures.

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 DI water, 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 DI water.
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 DI water, 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 DI water, sterile filtered aliqoted and stored at -20 °C.

List of used Strains

Coming soon.

List of used Primers

Coming soon.

E. Coli Culture Conditions

Coming soon.

E. Coli Continuous Cultivation

Coming soon.

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 pRhl und pLas autoinducers n-buturyl-homoserinlactone and n-oxododecanoyl-homoserinlactone 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 times 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

100 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 shock frozen in liquid nitrogen. The vials were stored at -80°C.

Cryopreservation

Coming soon.

Minipreparation of Plasmid DNA

Coming soon.

PCR

Colony PCR
ColonyPCR 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
PhusionPCR

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 and xxxV 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 out with a razor blade. 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 ca. 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. 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 S.O.C.-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 chemo competend 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

50ml of overnight culture were pelleted for 10 min at 3220 g. The supernatant was discarded and the cells were resuspended in 4ml 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 mins between the sonication cycles. The cell debris were pelleted for 5 mins at 16,100 g. The supernatant containing the soluble chromoproteins was sterile filtered (0.2µm) 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 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.

Imaging of Cells producing Chromoproteins

Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM n-buturyl-homoserinlactone (pRhl) or n-oxo-dodecanoyl-homosereinlactone (pLas) 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.

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 <div id="Proteinextraction" class="menuSection">
      <h2><a href="#Proteinextraction">Extraction of Chromoproteins</a></h2>
-     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Coming soon</p>
+     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">50ml of overnight culture were pelleted for 10 min at 3220 g. The supernatant was discarded and the cells were resuspended in 4ml of 1x Phosphate Buffered Saline (PBS).<br>
+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 mins between the sonication cycles. The cell debris were pelleted for 5 mins at 16,100 g. The supernatant containing the soluble chromoproteins was sterile filtered (0.2µm) and stored at 4 °C.
+</p>
   </div>

Team:Braunschweig/Protocols

From 2013.igem.org

Revision as of 23:55, 28 September 2013

Protocols

Recipes for Solutions and Media

List of used Strains

List of used Primers

E. Coli Culture Conditions

E. Coli Continuous Cultivation

Measurement of Growth Curves

Preparation of competent cells

Cryopreservation

Minipreparation of Plasmid DNA

PCR

Gel Electrophoresis

Gel Slice Preparation

Cloning

Electroporation

Extraction of Chromoproteins

Measurement of Absorption and Emission Spectra of the Chromoproteins

Imaging of Cells producing Chromoproteins

Detection of Autoinducers

Agar Diffusion Test

Evaluation of the leakyness of promotors

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