IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.2

Protocol

Status:

• Partially completed
• More Data Points needed

Aims:

• To create a series of dilutions of GFP using a purified sample of GFP

Equipment

Reagents

• Our Prepared S30 extract
• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Commercial S30 T7 extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl T7 S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• MiiA water x1ml
• GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)
• Prepared dilutions of AHL to 1mM

Reagents

• Recombinant GFP (Mw=27,000Da) Link to product information. It is in the following form;
• 100ug in buffer of 1mg/ml, this means in the solution there is:
  • 3.7x10^-9 moles of GFP
  • 0.037 M (moles dm^-3)
  • Volume supplied to us is 0.1mL

Protocols

• The moles of GFP that our in vitro systems is expected to produce is 7.4x10^-12 moles, this is based upon the quoted expected protein production of 200ng (2x10^-7/27000Da). If the total volume of the cell extract is 100ul, this means that the concentration is around 7.4x10^-8 M.
• We need a sufficient range of dilutions to cover this range. However, the relationship between fluorescence and molcules of GFP is linear at concentrations in uM and lower. Therefore, if we have a sufficient range we should be able to plot an accurate calibration curve.
• We propose covering the following range of 8 data sets with 2 repeats for each:
  

1.000uM
0.500uM
0.250uM
0.125uM
0.060uM