IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation/Calibrating Fluorescence

=Calibrating Fluorescence=

Summary
Protein quantification is not new to measuring relative fluorescence in cells, but for our experiment it would be a bonus to be able to relate the relative measurements to that of the actual molecule numbers per cell. This would facilitate comparison of results from the plate reader with measurements that would be taken across the various parameters as described (eg. Temperature change, for example). This process of calibrating GFP counts using the GFP-separated labels on the fluorometer plate reader to GFP concentration is adapted from protocols described by Endy Lab.

While calibration cannot be done directly to our experimental sample as the quantum efficiency of purified GFP in solution may be different from GFP in vivo, GFP from experimental samples to the GFP counts of lysed samples from the same original culture can be obtained by measuring GFP before and after cell lysis. Next, a standard curve that relates the concentration of purified GFP in non-fluorescent cell lysate to GFP counts can be measured. The combing of both sets of data would yield a calibration from GFP counts for experimental samples to GFP concentration.

[Insert image of calibration overview]

Experiment 1: Fluorescence vs extracellular [GFP] molecules
Known concentrations of GFP are diluted into a range of dilutions and are then mixed with cell lysate of a particular chassis.

Equipment

 * 1) Flurometer plate reader for measurement of fluorescence levels

Reagents
The gold standard for calibration experiments. Concentration of GFP in sample can be measured using a nanodrop and diluted to different concentrations using distilled water. Since GFP needs to be correctly folded we need to dilute the concentrated stock into a neutral pH buffer (TE).
 * 1) Purified GFP.

Experiment 2: Fluorescence vs Cultures of varying unknown intracellular [GFP]
Measuring the fluorescence of cultures of cells at set time intervals would measure a set of unknown intracellular [GFP].

Equipment

 * 1) Flurometer plate reader for measurement of fluorescence levels

Reagents
F2620-pSB3k3 in MG1655 grown in supplemented M9 media overnight and diluted back 1/500. Once the cell reach an OD of around 0.7 we induce 5ml of the culture by adding AHL to a concentration of 100nM. Every 10 mins for the next hour we induce another 5ml of the culture. This produces a set of cultures with different amounts of GFP per cell. The ODs of all the cultures were similar after the 60 mins. For each culture, add 3 replicates of 200μl to wells on a 96 well plate. Record the GFP counts and the absorbances of the culture. 1ml of each of the cultures was then lysed according to the method below. Cells not expressing GFP that were grown and lysed in an identical manner to the receiver cells were used to dilute the purified GFP.
 * 1) Experimental Samples.

Experiment 3: Fluorescence vs Cultures of varying unknown extracellular [GFP]
Lysing samples of Experiment 2 will allow the relation of an unknwon intracellular [GFP] to that of extracellular [GFP].

Equipment

 * 1) Flurometer plate reader for measurement of fluorescence levels
 * 2) 4oC Cold Room for lysis preparation
 * 3) Tabletop centrifuge for spinning down lysate
 * Ice

Reagents

 * 1) Experimental Samples
 * 2) Tris buffer
 * 3) B-Per II

Protocol

 * Cell Lysis
 * 1) Start with E. coli grown in supplemented M9 media to an OD of 0.86.
 * 2) Put Tris buffer at 4C.
 * 3) Incubate samples on ice for 10mins.
 * 4) Pipet 3 x 1ml replicates into 1.7ml eppendorf tubes.
 * 5) Centrifuge all samples at 4C. for 4mins at 13000rpm using a tabletop centrifuge.
 * 6) Remove supernatant.
 * 7) Resuspend the sample pellet in 500μl of chilledTris (10mM, 100mM NaCl, pH 8).
 * 8) Repeat steps 5 and 6.
 * 9) Resuspend cells in 50μl B-Per II.
 * 10) Add 1 unit Benzonase per 1ml of culture.
 * 11) Incubate at room temperature for 30mins.

This should lead to >90% lysis according to earlier plating experiments.

Solutions
For 1L of 1X Supplemented M9 media

200 mL 5X M9 minimal salts 34 mL 10 mg/mL thiamine 10 mL 40% glycerol 20 mL 10% Casamino acids 2 mL 1M MgSO4 100 &mu;L 1M CaCl2 733.9 mL H2O
 * 1) Dissolve 56.4 g Bacto M9 minimal salts, 5X from Difco in 1L H2O
 * 2) Separate into 200 mL aliquots
 * 3) Autoclave to sterilize. 121&deg;C for 15 minutes.
 * 1) Dissolve 10 mg per mL of H2O
 * 2) Use a 0.22 &mu;m filter to filter-sterilize
 * 1) Add 80 mL glyerol to 120 mL of H2O
 * 2) Autoclave to sterilize
 * 1) Dissolve 50 g Bacto Casamino acids from Difco in 500 mL H2O
 * 2) Autoclave to sterilize
 * 1) Dissolve 24.65 g MgSO4&middot;7H2O in 100 mL H2O
 * 2) Autoclave to sterilize
 * 1) Dissolve 14.7 g CaCl2&middot;2H2O in 100 mL H2O
 * 2) Autoclave to sterilize
 * 1) Sterilize deionized water in autoclave

Combine above solutions using sterile technique. (May notice some precipitation during preparation but precipitate should go back into solution once volume is brought up to 1L with sterile H2O.) Add antibiotic as appropriate and store at 4&deg;C

Calculations
More info to be added.