IGEM:IMPERIAL/2009/M0/Assays/1.2

=Glucose variation and time delay=

Aims

 * Characterise the tunable time duration it takes before GFP expression (M2 activation)

Assay
The cells will be grown until OD= 0.7.

The RFP value will be monitered, although it is the GFP values that are more critical in this assay.

OD and fluorescence data for GFP which can be converted in Specific Promoter Units (SPUs) by a calculator found on the Registry of Standard Biological Parts.

The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection for CRP promoter)

The IPTG concentrations will be taken from the previous experiment (see Determining concentration of IPTG)

Measurement phase

 * Spectrophotometer
 * Fluorimeter
 * 96 well plates

Assembly phase

 * Miniprep reagents
 * Ligation reagents
 * Transformation reagents

Media
M9 Minimal Media

Disodium Phosphate (12.0g)

Potassium dihydrogen phosphate (6.0g)

Sodium Chloride (1.0g)

Ammonium Chloride (2.0g)

Magnesium Sulphate (0.75g)

Glycerol (5.0g per L)= 54.39uM 0.5%

Secondary carbon source of choice (5g per L)   0.5% per 100ml

Glucose

Protocol
M9 Minimal Media Background:

Minimal M9 media is a defined minimal growth media suited to optical density based studies on account of its low absorbance and autofluoresence.

MgSO4 solution precipitates when heated and therefore cannot be autoclaved. To maintain sterility, MgSO4 solution should be filter sterilised (Minisart® 0.20µm syringe filter) and added after the other chemicals had been dissolved, mixed and autoclaved. To support bacterial growth, M9 medium requires the addition of a carbon source.

M9 Minimal Media Preparation:


 * Measure out the following reagents and dissolve them in 1000ml of sterile H20:

Disodium Phosphate = 6.0g

Potassium dihydrogen phosphate = 3.0g

Sodium Chloride = 0.5g

Ammonium Chloride = 1.0g

Glycerol (5.0g per L)= 54.39uM 0.5%

Secondary carbon source of choice (5g per L)


 * Repeat the step above such that a total of 2000ml of M9 minimal media is prepared.

IPTG standard solution Preparation:


 * 1g of IPTG dissolved in 40ml of dH2O (filter sterilize) to get 0.1M IPTG

Glass-ware Preparation:
 * 6 conical flasks

Other Protocols Needed

 * Miniprep
 * Ligation
 * Transformation

Assembly Instructions
1. Prepare the test promoter by miniprep of colonies containing promoter test construct followed by restriction digest with Xbal and PstI.(see miniprep protocol)

2. Prepare the GFP reporter device (BBa_E0240) by miniprep of miniprep of colonies containing E0240 followed by restriction digest with Xbal and PstI.

3. Prepare backbone plasmid (pSB3K3) by preparative PCR of pSB3K3-P1010 using primers BBa_G1000 and BBa_G1001 followed by restriction digest with EcoRI and PstI.



4. Combine the test promoter, GFP reporter device, and backbone plasmid in a 3-way ligation to build the promoter test construct. (see ligation protocol)



5. Prepare empty backbone vectors.

5. Transform the promoter test construct and empty vectors into TOP10 cells. Select for transformants on Kanamycin plates. Optimum DNA for each component could be approximately 10ng per microlitre.(see transformation protocol)



6. Measure the activity of the test promoter using the measurement instructions.



Promoter Measurement Protocol
1. Inoculate single colonies of E. coli cells into eppendorf tubes containing 1 ml of the pre-warmed (28°C) normal supplemented M9 medium with kanamycin (20 ug/ml). There will be 2 eppendorf tubes, one for cells containing the promoter test construct on the vector backbone, the other for control cells which are transformed with an empty vector.

2. Grow the cultures in the eppendorf tubes for approximately 6 hrs at 28°C with spinning at 70 rpm.

3. In the meantime, create 6 different media setups (10ml):

Flask 1: 0g  glc =  0mM Flask 2: 0.2g glc = 1.11mM Flask 3: 0.4g glc = 2.22mM Flask 4: 0.6g glc = 3.33mM Flask 5: 0.8g glc = 4.44mM Flask 6: 0.8g glc = 4.44mM

4. Dilute the cultures 1:100 into 10 ml of fresh media and grow the cultures

5. Use an automatic plate reader to detect the time at which the culture reaches an OD of 0.7.

6. After the OD reaches 0.7, add a certain volume of 0.1M IPTG to 10ml of culture into each conical flask setup and mix well. (for amounts, see assay on determining IPTG concentration)

7. Transfer 200 ul aliquots from each culture into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner). (for a better estimate of timing, refer to assay on determining IPTG concentration)

8. Incubate the plate in a multi-well fluorimeter (Perkin Elmer) at 28°C and assay with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (Excitation peak: 584 nm  Emission peak: 607 nm, 0.1 seconds, CW lamp energy 12901 units), and shaking (3 mm, linear, normal speed, 15 seconds). (Just measures GFP – fluorescence over time).

9. Repeat the absorbance and fluorescence measurements every hour during mid-exponential growth for at least 5 hours.

10. Take readings every half hour approximately from the time GFP is synthesized. (For a rough estimate, see assay on determining IPTG concentration)

8. Determine background absorbance by measuring wells containing only media. This should be subtracted from subsequent absorbance readings.(Blank).

9. Determine background fluorescence at different ODs from the fluorescence of control cells without a GFP expressing vector. (This is from the culture containing the untransformed cells). (Control)

Wet Lab Protocol:
PROTOCOL:

Day 1:

M9 Media Preparation:


 * Measure out the following reagents and dissolve them in 500ml of sterile H20:

Disodium Phosphate = 3.0g

Potassium dihydrogen phosphate = 1.5g

Sodium Chloride = 0.25g

Ammonium Chloride = 0.5g

Secondary Carbon Source = 2.0g


 * Label 10 falcon tubes as shown below (indicates glucose content):

1) 1.000 g

2) 0.500 g

3) 0.250 g

4) 0.125 g

5) 0.063 g

6) 0.031 g

7) 0.016 g

8) 0.008 g

9) 0.004 g

10) 0 g


 * Decant 50ml of M9 minimal media into each of the 10 falcon tubes
 * Measure out and add the appropriate amount of glucose as indicated by each of the tube labels.
 * Autoclave the M9 media to ensure sterility.

Starter Culture Preparation:


 * Look for GFP producing colony on a plate of transformed colonies.
 * Innoculate a falcon tube filled with 10ml of LB agar with a transformed colony.
 * Incubate overnight at 37 degrees centigrade.

Day 2:


 * Dissolve 0.25g of Magnesium Sulphate in 20ml of H20.
 * Using a Minisart® 0.20µm syringe filter, add 2ml of Magnesium Sulphate solution to each falcon tube.

Day 3:


 * Place starter culture on ice and measure remaining glucose content (using Biovision glucose assay kit - see Assay M0 0.1).


 * Innoculate each falcon tube with 1ml of starter culture.


 * Incubate cultures at 37 degrees.


 * Every 20 minutes, remove 1ml aliquotes (into eppindorfs) and place on ice. Also measure OD (at 600nm at sample points).


 * After final sample collection, transfer samples to a 96 well plate and measure fluoresence output.