IGEM:MIT/2007/Notebook/2007-7-30

Mer-GFP Expression Protocol

1. Mix Diluted LB Media (LB4.10)
   1. 4g NaCl, .5g Yeast Exract, 1g Tryptone + 1L H20
2. Autoclave LB4.10
3. Grow up 10 mls ON at 30°C
4. Dilute back 1:100 in fresh LB4.10 (50 ul to 5 ml)
5. Add HgCl2 in following concentrations (ng/ml):
   1. 0
   2. 5
   3. 10
   4. 25
   5. 50
   6. 75
   7. 100
   8. 150
   9. 300
   10. 500
   11. 1000
   12. 10000
6. Grow at 30°C for 16h
7. Wash a 3ml sample of each (twice) and resuspend in .9% NaCl (Minimizes Background)
8. Measure flourescence

• Excitation wavelength - 395nm
• Emission wavelength - 509nm

Measure out 0.1 g and dilute in 10 mL water This will give you 10 mL of 10 mg/mL Take 100 uL and dilute in 10 mL water This will give you 10 mL of 100 ug/mL Take 100 ul and dilute in 10 mL water This will give you 10 mL of 1 ug/mL

Live Cell Immunoassay (Eric)

• Today's assay - fluorescent staining of T7 tag engineered into the CPX insert. Checks for expression of CPX and polystyrene-binding peptide

1. Induce cells with current conditions
2. Spin down 1 OD cells
3. Resuspend in 1 mL PBS-BSA (5 mg/mL) -- make 50 mL
4. Spin down 0.2 OD (200 µl); aspirate off media
5. Resuspend in 100 µl 1:500 (experiment?) antiT7 antibody in PBS-BSA (1 mL PBS-BSA; 2 µl antiT7)
6. Incubate on ice for 30 minutes
7. Add 800 µl cold PBS-BSA
8. Spin down cells and remove media
9. Resuspend in 100 µl 1:20 anti-mouse antibody in PBS-BSA (1 mL PBS-BSA; 50 µl anti-mouse)
10. Incubate on ice for 30 minutes IN THE DARK
11. Repeat Step 7
12. Repeat Step 8
13. Repeat Steps 7 and 8; wash
14. Resuspend in 100 µl PBS-BSA
15. Look under fluorescent microscope

• Do induced + controls

Results of Immuno-Stainng Assay

• 

  AHL-6 1

• 

  AHL-6 2

• 

  AHL-6 3

• 

  AHL-7 1

• 

  AHL-7 2

• 

  AHL-7 3

• 

  AHL-8 1

• 

  AHL-8 2

• 

  AHL-8 3

• 

  Control 1

• 

  Control 2

• 

  Control 3