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From 2014.igem.org

6^th June

• We continue our experiments to have BL21 without plasmid.


• Preparation of LB + Amp and LB + Chlo plates.

A₆₀₀ preculture VPGXG-20 = 2,460
A₆₀₀ preculture VPGXG-40 = 2,678
-> Culture of VPGXG-20 and VPGXG-40 at 37°C

T=0, A₆₀₀ VPGXG-20 = 0,139
A₆₀₀ VPGXG-40 = 0,072
-> IPTG induction when A600 = 0,8 and culture at 25°C

• Collection of cell lysate


1. Pour ELP cultures into 1L plastic centrifuge bottles
2. Centrifuge 15min at 3,000 rpm and 4°C
3. Discard supernatant into 4L flask and bleach before disposal in the sink
4. Repeat the latter steps if desired to consolidate cultures
5. Resuspend cell pellet in 20mL PBS
6. Transfer cell solution to a 50mL Falcon tube for sonication
7. Lyse cells by sonication on ice (3min: 10s on / 20s off – let sit on ice then repeat for a total of 2 sonication cycles)
8. Transfer lysate into clear centrifuge tubes


• ELP purification by Inverse Transition Cycling (ITC)
1. Add 10% polyethylenimine (PEI) (2mL per 1L culture) and gently mix
2. Centrifuge at 4°C and 14,000 rpm for 15min
3. Decant supernatant into clean centrifuge tube and discard pellet
4. Allow solution to warm, add NaCl (not exceeding 3M) and gently mix - The solution should turn turbid with the ELP transition
5. Centrifuge immediately after the ELP transition at room temperature and 14,000 rpm for 15min (HOT SPIN 1)
6. Discard supernatant and resuspend ELP pellet in cold PBS(2mL per 1L culture)
7. Transfer solution to 1.5mL labeled micro-centrifuge tubes
8. Put tubes on ice 30min
9. Centrifuge at 4°C and 14,000rpm for 10min (COLD SPIN 1)
10. Decant or pipette supernatant into clear micro-centrifuges tube and discard pellet
11. Allow the solution to warm and if necessary add 5M NaCl solution until ELP transition occurs
12. Centrifuge at 37°C and 14,000rpm for 10min (HOT SPIN X)
13. Discard supernatant, add cold buffer, and resuspend pellet
14. Centrifuge at 4°C and 14,000rpm for 10min (COLD SPIN X)
15. Decant or pipette supernatant into clear centrifuge tube and discard pellet
16. Perform 3-5 rounds of ITC purification

Today we stop protocol after resuspend cell pellet in 20mL PBS and store at -80°C.