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From 2010.igem.org

Contents 1 Andreas 1.1 Growth curve assay 1.2 Removal of insertion in BioBrick suffixes 1.2.1 Plasmid prep 2 Nina 2.1 Protein purification 2.1.1 column equilibration 3 Mimmi 3.1 SOD activity assay

Andreas

Short day in lab - birthday celebrations!

Growth curve assay

Inoculated fresh LB with 0.3 mM IPTG with ON cultures to a startup OD of 0.2 and a total volume of 50 ml in 150 ml side-arm E-flasks.

Was advised to use a smaller cultures for the flask size. Experiment canceled after about an hour; will be repeated next week.

Removal of insertion in BioBrick suffixes

Plasmid prep

Spun down pSB1C3.SOD ON culture at 13,000 x g, 10 min. Supernatant discarded and cell pellet stored in -20 °C for later plasmid prep.

Nina

Protein purification

I performed a protein purification of SOD.His (N terminal). The pellet came from an IPTG induced 12 ml overnight culture.

Lysis buffer:

630 ul * 2 = 1260 ul lysis buffer

Wash buffer:

10X lysis buffer 12.6 ml

Elution buffer:

5X lysis buffer 6.3 ml

• PMSF

100 * volume = 1260 * 1

12.6 ul PMSF added in lysis buffer

• Imidazole

2 * volume = 1260 * 10*10^-3

6.3 ul imidazole added in lysis buffer

• Imidazole

2 * volume = 1260 * 20*10^-3

126 ul imidazole added in wash buffer

• Lysozyme

The tip of a spoon was added in lysis buffer

• DNase

20 ug/ml was added in lysis buffer

column equilibration

The work is carried out in a cold room.

• Ni-resin was vortexed and 1 ml was added in a drop column.
• 5 ml wash buffer was added and run through the column (to equilibrate the Ni-resin).
• The lysis mixed with the bacteria pellet was added in the column. However, the mixture seemed to plug the column and nothing could run through.

I left this in the cold room over the weekend to check with anyone at the department what to do and if I still can work with this material.

Mimmi

SOD activity assay

• Searching "web of science" for SOD activity assays

• Generates O₂^-
  • Xanthine + xanthine oxidase
  • NADH + phenazine methosulphate
  • riboflavin + methionine

• Reacts with O₂^- and can be measured
  • cytochrome C
  • nitro-blue tetrazodium NBT
  • adrenaline
  • pyrogallol
  • hydroxylamine

• Catalase-like test?
  • No, since E. coli are catalase positive and SOD differences will not be detectable

• SOD activity test that chemically produces O₂^-

mix
graded levels of SOD	?	Incubate in RT, darkness for 10min Measure A540 negative control = without NADH
NBT	1mg
phenazine methosulphate	10µg
EDTA	1µM
gelatin	1mg
phosphate	0.1M
NADH 1mM	0.1ml
tot	3ml
pH=7.8