From 2013.igem.org

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Revision as of 18:42, 25 September 2013

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

Coming soon.

Cryopreservation

Coming soon.

Minipreparation of Plasmid DNA

Coming soon.

PCR

Colony PCR, Phusion PCR coming soon

Gel Electrophoresis

Coming soon

Gel Slice Preparation

Coming soon

Cloning

Coming soon

Electroporation

Coming soon

Extraction of Chromoproteins

Coming soon

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.

@@ Line 104: / Line 104: @@
 <b>Clavulanic acid</b><br>
-g Clavulanic acid was dissolved in 100 mL Ethanol, sterile filtered aliqoted and stored at -20 °C.
+g Clavulanic acid was dissolved in 100 mL DI water, sterile filtered aliqoted and stored at -20 °C.
 </p>

Team:Braunschweig/Protocols

From 2013.igem.org

Revision as of 18:42, 25 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