IGEM:IMPERIAL/2009/M0/Assays/1.1a

=Choice of secondary carbon source=

Aims

 * To measure the activity of the CRP promoter under each different carbon sources, and consequently choosing the most effective secondary carbon source.


 * In addition, we can also characterise the CRP promoter under the most effective secondary carbon source.

Assay
Monod classified sugars into class "A" or "B". Class A sugars (mannose, fructose, mannitol) do not give rise to diauxic growth when present in the medium with glucose. By contrast, class B sugars (arabinose, maltose, rhamnose, lactose, xylose, galactose) do give rise to diauxic growth when present with glucose.

The experiment will generate OD and fluoresence data which can be converted in Specific Promoter Units (SPUs) by a calculator found on the Registry of Standard Biological Parts.

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%

Glucose (0.1g per L) = 556uM 0.01% per 100ml

Galactose (0.5g per 100mL) = 27.7mM   0.5% per 100ml

Maltose (0.5g per 100mL) = 14.6mM  0.5% per 100ml

Arabinose (0.5g per 100mL) = 33.3mM   0.5% per 100ml

Xylose (0.5g per 100mL) = 33.3mM   0.5% per 100ml

Rhamnose (0.5g per 100mL) = 30.5mM  0.5% per 100ml

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


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

Glycerol (5.0g per L)= 54.39uM

Glucose (0.1g per L) = 556uM

Glass-ware Preparation:


 * Label a 24 well plate with 10 wells as shown below:

1) Galactose (Transformed)

2) Maltose (Transformed)

3) Arabinose (Transformed)

4) Xylose (Transformed)

5) Rhamnose (Transformed)

6) Galactose (control-empty vector)

7) Maltose (control-empty vector)

8) Arabinose (control-empty vector)

9) Xylose (control-empty vector)

10) Rhamnose (control-empty vector)


 * Decant 100ml of M9 minimal media into each of the 10 conical flasks.
 * Measure out and add 0.5 grams of each carbon source and add it to the conical flask with the corresponding label.
 * Autoclave the M9 media to ensure sterility.
 * Measure out and dissolve 0.75g of Magnesium Sulphate in 30 ml of H20.
 * Using a Minisart® 0.20µm syringe filter, add 2 ml of Magnesium Sulphate solution to each conical flask.

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 a 24 well dish containing 1 ml of the pre-warmed (28°C) normal supplemented M9 medium with kanamycin (20 ug/ml). There will be 2 wells, 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 24 well dish for approximately 6 hrs at 28°C with spinning at 70 rpm.

3. Use an automatic plate reader to detect the time at which the culture reaches an OD of 0.7. (or other value as long as the amount of atarting culture is fixed)

4. In the meantime, create your 10 setups with 1ml of media in a 24 well plate with different carbon sources.

1) Galactose (Transformed)

2) Maltose (Transformed)

3) Arabinose (Transformed)

4) Xylose (Transformed)

5) Rhamnose (Transformed)

6) Galactose (control)

7) Maltose (control)

8) Arabinose (control)

9) Xylose (control)

10) Rhamnose (control)

5. Add 10ul of culture into 1 ml of the 10 setups above and grow the cultures under the previous conditions.

6. After about 3 hours, 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 (485 nm excitation filter, 525 nm emission filter, 0.1 seconds, CW lamp energy 12901 units), and shaking (3 mm, linear, normal speed, 15 seconds). (Just measures GFP – fluorescence over time).

8. Repeat the absorbance and fluorescence measurements every half an hour until GFP fluorescence plateaus off.

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)