Team:Goettingen/Notebook/evolution July

Overexpression of proteins in E. coli

13.07.18

  1. Preculture over night at 37°C with agitation has to be prepared.
  2. Inoculate 4x 0.5 L LB medium with ampicillin in 2 L shake flasks to an OD600=0.05.
  3. At an OD600=0.6 add an inducer, like IPTG.
  4. Incubate the cultures for 3 h at 37°C with agitation.
  5. Harvest the cells with centrifugation for 10 min at 4°C and 5,000 rpm. Discard the supernatant.
  6. Resuspend the pellet in 25 mL buffer W (cold) and transfer the samples into 50 mL falcon tubes.
  7. Centrifuge the samples for 10 min at 4°C and 5,000 rpm. Discard the supernatant and store the pellet at -20°C.

Cloning of biobricks 2, 9, 10, 12 and overexpression of AroA and AroE

19.07.18

The digest and ligation reactions were performed as described before. Furthermore, a purification of the overexpressed proteins AroE from B. subtilis and AroA from E. coli was performed as decribed in the following paragraph.

  1. The AroA strain was cultured in 2 L medium, the AroE strain in 1 L as described before. Harvest the cells with centrifugation at 20,000 rpm for 20 min at 4°C.
  2. Resuspend the pellet in 15 mL buffer W (ice-cold!).
  3. Disrupt the cells with the French press. Repeat this step 2 times.
  4. Centrifuge the samples 30 min at 17,500 rpm and 4°C.
  5. Prepare the 10 mL column by BioRad with 1 mL Strep-Tactin Sepharose which equals a column volume of 500 µL.
  6. Load the supernatant from the centrifugation on the column. Save the flowthrough and load it again on the column.
  7. Wash the column with buffer W: 1. run: 5 mL. 2. run: 1.25 mL.
  8. Elute the protein with buffer E. Load ½ of column volume on the column. Save the flowthrough.
  9. Elute the protein with buffer E two times. Load the column volume on the column. Save the flowthrough.
  10. Load the samples on a SDS protein gel. Mix 15 µL sample and 5 µL Laemmli buffer (5×) and boil it for 10 min at 95°C.
  11. Perform a Bradford assay with the elution fractions.

The results of the protein purification are shown in the next figure.

<img src="T--Goettingen--Notebook_1_190718.png"> 
                   <img src="T--Goettingen--Notebook_2_190718.png">

The SDS page from the protein AroE from B. subtilis is shown in this figure.

The SDS page from the protein AroA from E. coli is shown in this figure.

The Bradford assay was performed as described in the Experiments section. For AroE, 4 µL and 8 µL were used and the concentration of AroE sample 1 was determined as 2.5 µg/µL and in the second sample as 2.03 µg/µL. The concentration for AroA was determined using 10 µL and 14 µL as 0.15 µg/µL.