Team:Goettingen/Notebook/evolution June

01.06.18

100 µl of the SP1 over night cultures were inoculated in 10 ml CS-glucose medium (without trp) at 37°C.

IGEM35-44 were streaked out on CS-glucose agar plates containing 40 mM glyphosate.

   <img src="T--Goettingen--Agarplatten_310518.png">

Two plates of CS-glucose with 10 mM glyphosate were prepared and BP236 was inoculated on these plates at 37°C for multiple days.

A plate reader assay was performed. 5 µl of a SP1 culture were used to inoculate 100 µl of CS-glucose medium containing different glyphosate concentrations. The glyphosate concentrations were 0 mM, 0.5 mM, 1 mM 1,5 mM 2 mM 2.5 mM, 3 mM and 3.5 mM. It was worked with triplicates of every concentrations.

04.06.18

For the disc diffusion assay, the BP236 mutants were inoculated in LB medium, then washed with 1x S-salts and plated on CS-glucose medium as previously described. A disc was placed in the middle of the plates and 5 µl glyphosate were placed on the disc.

For a plate reader assay, SP1 and 168 were inoculated in liquid LB medium at 28°C over night.

BP236 mutants were harvested and the pellets were frozen at -20°C.

05.06.18

The plate reader assay was repeated with SP1 and 168 as described above.

The disc diffusion assay was evaluated.

Inoculation of over night cultures of BP233 and BP235 at 37°C and 220 rpm.

06.06.18

Plating of BP233 and BP235 on CS-glucose plates containing 0, 10, 20, 30, 40, 50 and 60 mM glyphosate

400 µl of the over night cultures of BP233 and BP235 were inoculated in 10 ml LB medium and grown at 37°C for 4 hours.The cells were harvested and washed with C-salts as described previously. 100 µl of the cells were plated on CS-glucose plates containing 0, 10, 20, 30, 40, 50 and 60 mM glyphosat

11.06.18

BP 235 mutants from the 50 and 60 mM glyphosate CS-glucose plates were picked and stuck out again on 50 and 60 mM glyphosate CS-glucose plates. The plates were incubated at 37°C.

Inoculation of over night cultures of WT 168 and SP1 in 4 ml LB.

12.06.18

The E. coli strain XL1-Red was transformed with pIGEM2 according to the following steps.

  1. Prewarm SOC medium to 42°C
  2. Thaw the XL1-Red competent cells on ice
  3. Alliquot 2x100 µl cells in 1.5 ml reaction tubes
    4. +
  4. Add 1.7 µl β-mecaptoethanol
  5. Swirl the contents of the tube gently, incubate the tube on ice and swirl every two minutes
  6. Add 0.5 µl pIGEM2 to one reaction tube and leave the other one without DNA as a negative control
  7. Let rest on ice of 30 min
  8. Heat shock the reaction tubes at 42°C for 45 sec
  9. LEt the tubes rest on ice for 2 min
  10. Add 0.9 ml prewarmed SOC medium to the cells and let the tubes shake for 1 h at 37°C
  11. Plate 100 µl of the cells on agar plates containing the appropriate antibiotic and then centrifuge the tubes for 1 min at 13000 rpm. Resuspend the pellet in 50 µl medium and plate.

100 µl of the over night cultures of 168 and SP1 were transferred to 10 ml CS-glucose medium. The cultures were incubated at 37°C and 220 rpm for 4 h. The plate reader assay was performed as described previously.

over ngiht cultures were prepared of 168 and SP1 for a plate reader assay the following day.

13.06.18

A plate reader assay was performed with 168 and SP1 in the same manner as previously described.

The colonies of the 50 colonies of the XL1-Red transformation plates (12.06.18) were picked and incubated over night at 37°C in 4 ml LB Amp.

14.06.18

10 cultures were pooled in 15 ml LB Amp, 100 µl of each culture.

15.06.18

Plasmid extraction using the Macherey and Nagel kit. The DNA was eluted in 50 µl water and the individual DNA concentrations were measured using the nanodrop.

Culture DNA concentrations in ng/µl
1 111.7
2 117
3 191.5
4 96.3

Singularization of BP235 mutants

18.06.18

Three isolated BP235 mutants that were grown on 40 mM glyphosate, were isolated on LB-Medium plates and incubated over night at 37 °C. After Incubation, the different singularized strains were cultivated and used for further characterization.

Transformation of SP1 with pIGEM2

25.06.18

The transformation of SP1 was performed as described <a href="https://2018.igem.org/Team:Goettingen/Experiments">here</a>. After incubation of the specific selection plates containing erythromycin and lincomycin, the resulting colonies were counted. The 5 plates that show growth of several colonies were stamped on CS-Glucose plates with 10 mM glyphosate. The plates were also incubated at 37 °C.

Singularization of stamped colonies

29.06.18

After incubation for 3 days at 37 °C the stamped colonies were singularized on CS-Glucose plates that contain tryptophane and 10 mM glyphosate. 7 different colonies and the wildtype SP1 were singularized on each plate.