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From 2010.igem.org

Contents 1 Andreas 1.1 Growth curve assay 1.1.1 Induction 1.1.2 OD590 measurements 1.1.3 ON cultures 2 Nina 2.1 Protein purification 2.1.1 Lysis 2.2 PCR Colony

Andreas

Growth curve assay

New attempt to measure growth curves for CPPs.

Induction

• 150 ml side-arm E-flasks
• 15 ml LB
• 150 μl ON culture (1:100)
  • BL21
  • ON cultures set by Mimmi 13/10
• 37 °C, 220 rpm

OD₅₉₀ measurements

	0 min	30 min	60 min	90 min	120 min	150 min	180 min	210 min	240 min	270 min
pEX.SOD⋅His	0.03	0.04	0.10	0.23	0.45	0.78	1.06	1.36	1.56	1.67
pEX.nTra10⋅SOD⋅His	0.03	0.04	0.09	0.19	0.36	0.61	0.94	1.18	1.42	1.56
pEX.nTAT⋅SOD⋅His	0.03	0.05	0.10	0.23	0.49	0.78	1.06	1.35	1.55	1.66
pEX.nLMWP⋅SOD⋅His	0.04	0.05	0.11	0.25	0.52	0.88	1.14	1.40	1.60	1.69

Experiment was canceled after 270 min as I was made aware that the spectrophotometer only makes accurate measurements up to ≈OD₅₉₀ = 0.5.

ON cultures

New ON cultures set (including two for Mimmi)

• 5 ml LB + Amp 100; 37 °C, 225 rpm
  • BL21, pEX.SOD⋅His
  • BL21, pEX.nTra10⋅SOD⋅His
  • BL21, pEX.nTAT⋅SOD⋅His
  • BL21, pEX.nLMWP⋅SOD⋅His
  • (BL21, pEX.SOD)
  • (BL21, pEX.yCCS)

Nina

Protein purification

Lysis

I have talked to people in the department that work with protein purifications and they recommended me to start working from a bigger culture such as 40 ml instead of 12 ml.

I used a 40 ml SOD.His (N terminal) culture for this purification.

I performed a batch purification of SOD.His tagg in E.coli BL21. For this I used a 50 ml falcon tube as the column.

• Ni-NTA tube was vortexed to have a homogenized solution of Ni.
• 10 ml Ni-NTA was added in the 50 ml falcon tube (column).
• Centrifuged 30 sec at 4000 rpm to remove the supernatant the Ni was dissolved in.
• Added 30 ml lysis buffer without imidazole, DNase and PMSF for equilibration of the Ni-resin. Left on shake in 4 °C for 1 h.
• I prepared the lysis buffer:

For 5 ml pelleted culture use 630 ul lysis buffer

40 ml culture/5 = 8

8 * 630 ul lysis buffer = 5 ml

I double this 5 to 10 ml since I heard at the department that this would be good and there is no harm in using to much of lysis buffer.

Therefore I had 10 ml lysis buffer.

PMSF:

10000 ul/100 = 100 ul (1 mM PMSF) add in lysis buffer

Imidazole:

(10000 ul * 10*10^-3)/2 = 50 ul (10 mM Imidazole) add in lysis buffer

DNase:

10*10^3 ug/ml * Volume = 20 ug/ml * 10 ml

Volume = 20 ul (20 ug/ml) add in lysis buffer

• Mixed the lysis buffer with the pellet from 40 ml culture & transfered to a glas tube to sonicate for 5 min (75%)
• Transfered the sonicated lysate back to a falcon tube and centrifuged for 20 min 8000 rpm in the cold room.
• performed a ultracentrifugation on the supernatant 40 min 50000 rpm (1 ml of supernatant in each 10 centrifugation tubes with the size of 1 ml). I saved the pellet from both centrifugation steps. The first centrifugation removes the unopen cells and the second centrifugation pellets the membranes and debris from the first centrifugation.
• Centrifuged the column and removed the supernatant that had equilibrated the resin.
• Added the supernatant from the second centrifugation (50000 rpm) to the Ni-resin in the falcon tube. Incubated in 4 °C overnight on shake.

PCR Colony

I did a PCR colony screen on the transformations from yesterday.

36 tubes

Master Mix:

• MgCl2 18 ul
• Phusion buffer 5X 180 ul
• dNTP 18 ul
• primerF 54 ul
• primerR 54 ul
• polymerase 18 ul
• water 540 ul

Had 24 ul of the mix in each 36 tubes.

Elongation time for protein A samples: 45 sec and for fusion samples: 2 min.