Team:Groningen/Notebook/8 July 2009

From 2009.igem.org

Dry

A start was made with modelling arsenic uptake on the Modelling page. It was mentioned by Martijn (iGEM team member of last year) that there exists software to give an estimate of binding affinities, in case we can't find specific ones (he hasn't been able to find the right link yet though).

Also, a planning for Construction 1 was made, focussing on arsenic for now. During the meeting the lab workers agreed that apart from arsenic the next metals to look at would be Zinc and Copper.

Wet

Purification of HmtA (PA Q9I147 6xHis Tag)

Growing cells and purification

• Resuspend pellet in 50mM Kpi pH7,0 with 1M NaCl
• Spin down for 25 min at 80 000 rpm
• Add 1 ml solubilazation buffer to the pellet
  • Solubilzation buffer (for 25ml)
    • 1,25 ml Kpi (1M)
    • 2 ml NaCl ( 5M)
    • 6,67 ml Gly (75%)
    • 15,08 ml water
    • 30 min on ice
• Spin down for 25 min at 80 000 rpm
• Wash the Ni-NTA beads;
  • 30 ul Ni-NTA in 1 ml buffer A,
• Spin 2 min 3500 rpm, remove supernatant
• Add supernatant of protein to epi with the Ni-NTA pellet
• Incubate for 2 h, shaking
• Spin 4 min at 3500 rpm with table spin
• Wash
  • 2x with 1ml buffer B+0,1% triton (20mM imidazole)
    • store the supernatants for use in SDS-gel electrophoresis
  • 2x with 1ml bufferB+01,% Triton (40 mM imidazole)
    • store the supernatants for use in SDS-gel electrophoresis
• Elute
  • 3x with 30uL BufferC+0,1%Triton (500mM imidazole)
    • store the supernatants for use in SDS-gel electrophoresis