IGEM:IMPERIAL/2009/M0/Assays/1.1

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Assay 1.1

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.


Note, due to the large number of secondary (class B) carbon sources that have to be tested, it is reccomended that the experiment be split up either temporally or between multiple individuals.

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.

Equipment

Measurement phase

  • Spectrophotometer
  • Fluorimeter
  • 96 well plates

Reagents

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)


Glucose (8.0g)

Galactose (8.0g)

Maltose (8.0g)

Arabinose (8.0g)

Xylose (8.0g)

Rhamnose (8.0g)

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.


Glass-ware Preparation:

  • Label 15 conical flasks as shown below:

1) Glucose (Transformed)

2) Galactose (Transformed)

3) Maltose (Transformed)

4) Arabinose (Transformed)

5) Xylose (Transformed)

6) Rhamnose (Transformed)

7) No-carbon source (Transformed)

8) Glucose (Un-transformed)

9) Galactose (Un-transformed)

10) Maltose (Un-transformed)

11) Arabinose (Un-transformed)

12) Xylose (Un-transformed)

13) Rhamnose (Un-transformed)

14) No-carbon source (Un-transformed)

15) No-carbon source (Falcon Stock Solution)



  • Decant 100ml of M9 minimal media into each of the 15 conical flasks
  • Measure out and add 0.4 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.

Assembly.jpg

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)

Assemblyy2.jpg

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

Assemblyyy.jpg

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

Assemblyyyy.jpg


Promoter Measurement Protocol

Note: This test will be carried out for each secondary carbon source. The supplemented M9 minimal media will be prepared for each carbon source (as stated above) and the required one used for each test.

1. Inoculate single colonies of E. coli cells containing the promoter test construct on the vector backbone, into three 17 mm test tubes containing 5 ml of the selected pre-warmed (37°C) supplemented M9 medium with kanamycin (20 ug/ml). Note: The M9 media previously prepared will contain different carbon sources. Select the appropriate one for the carbon source you wish to test).

2. Grow the cultures in the 17 mm test tubes for approximately 20 hrs at 37°C with spinning at 70 rpm.

3. Dilute the cultures 1:100 into 5 ml of pre-warmed fresh media and grow the cultures for approximately 4 hours under the previous conditions (17 mm tubes, 37°C, spinning at 70 rpm).

4. After 4 hours, measure the OD600 of a 500 ul aliquot from each culture on a WPA Biowave Spectrophotometer. This will tell you how many cells you have.

5. Based on this OD measurement, dilute the cultures to the same OD (0.07) in 5 ml of pre-warmed fresh media and grow for one hour at 37°C. Here we try to make sure everything is at the same OD.

6. Transfer three 200 ul aliquots from each culture into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner). For ease of measurement.

7. Incubate the plate in a multi-well fluorimeter (Perkin Elmer) at 37°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. 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)

10. Measurements were taken from an approximately 30 min period in mid-exponential growth.

References

  1. Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, and Endy D. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng. 2009 Mar 20;3:4. DOI:10.1186/1754-1611-3-4 | PubMed ID:19298678 | HubMed [promoter1]