IGEM:IMPERIAL/2009/M2/Assays/2.1

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Assay 2.1: Can the chassis produce colanic acid?

While there are multiple strains of E.coli K12 that can produce colanic acid, it has been noted in the literature that some cannot. It is important that before selecting our K12 chassis, we ensure that it can produce colanic acid. Assay 2.1 has both a Dry Lab and Wet Lab component.


Dry Lab: Use NCBI to determine whether the colanic acid biosynthetic genes are present in the available chassis.

Vincent 05:11, 17 August 2009 (EDT): results of the search for: Top10, XL1 Blue, BL21 DE3 ?


BL21 DE3 = Yes

Top10 = Yes

XL1 Blue = ?

Wet Lab Protocol:

Reagent Source:

Object Where to buy Description Quantity Product Number Price
p-Fluoro-L-phenylalanine http://http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=F4646%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC p-Fluoro-L-phenylalanine 100 milli grams F4646 £43.40


CALCULATIONS:

p-Fluoro-L-phenylalanine (FPA) is required for the chemical induction of colanic acid biosynthesis.

FPA molar mass = 183.18

Equations = Amount(moles) x Molar Mass (grams/mole) = mass (grams)

According to the literature, agar plates should contain 5 X 10-6 M FPA for the induction of colanic acid biosynthesis.

According to the protocol, we require a 100ml stock solution to make the agar plates.

Therefore this stock solution should contain 5 X 10-7 moles of FPA.

Since the FPA molar mass is 183.18 grams per mole, we require 0.09159 mg of FPA for the protocol.


EQUIPMENT REQUIRED:

  • Sterile petri dishes (x6)
  • Marker Pen (x1)
  • Waterbath (x1)
  • Conical Flasks (at least 200ml volume) (x3)
  • Autoclave (x1)
  • Falcon tube (at least 20ml volume) (x3)
  • Sterile Strippette (x1)
  • Strippette pump (x1)
  • Incubator (x1)
  • Gilson Pipette p1000 (x1)


REAGENTS REQUIRED:

  • LB liquid media powder (2.5 grams)
  • LB agar media powder (3.7 grams)
  • p-fluorophenylalanine (0.09159 milli grams)
  • Distilled H20 (300 ml)


BIOFORMS REQUIRED:

E.coli Strains:

  • Top10
  • XL1 Blue
  • BL21 DE3


PROTOCOL:


Day 1:


Agar Plate Preparation:

  • Label one conical flask 'Flask A' and and a second 'Flask B'.
  • Fill 'Flask A' and 'Flask B' with 100ml of distilled H20.
  • Weigh out and add 3.7 grams of LB agar powder to each flask.
  • Measure out 0.09159mg of p-fluorophenylalanine and add to 'Flask B'.
  • Autoclave 'Flask A' and 'Flask B' to sterilise the media.


Innoculum Preparation:

  • Label a conical flask 'Flask C'.
  • To Flask C, add 100ml of H20 and 2.5 grams of LB powder for liquid media.
  • Autoclave all media to sterilise.
  • Using a sterile strippete, fill three falcon tubes each with 20ml of liquid LB media.
  • Label the three falcon tubes: 'Top10', 'XL1-Blue' and 'BL21 DE3'.
  • Select one colony from each strain plate and using a loop, transfer a single colony to the corresponding falcon tube.
  • Incubate the tube for 24 hours at 37 degrees centigrade. Successful innoculation is shown by a gradual clouding of the media.


Day 2:

Morning:

Plate Preparation:

  • Melt agar in 'Flask A' and 'Flask B' in a microwave.
  • Once melted, place the flasks in a water bath set to 50 degrees centigrade.
  • Label 6 petri dishes as shown below:

Image:ColanicAssay.JPG

  • Use the contents of 'Flask B' to make up the three plates marked '+ inducer'.
  • Use the contents of 'Flask A' to make up the three plates marked control.
  • Allow the agar to solidify.
  • Once solidified, store the plates in a cold room.


Afternoon:

Plating of cells:

  • Remove the innocula prepared on Day 1 from the incubator.
  • Using a P1000, pipette 1ml of cell culture (from the corresponding strain) onto each of the appropriatly labelled plates. E.g. 1ml of Top10 culture should be pippetted onto each of the two agar plates labelled 'Top10'.
  • After adding the innoculum, use a sterile spreader to obtain an even distribution on the surface of the plate.
  • Finally, incubate innoculated plates at 30 degrees centigrade for 60 hours.
  • Save the remaining inoculum (starter culture) for use in Assay 2.2.


Post 60 hour incubation:

  • Remove cells from the incubator and use visual analysis to identify mucoid phenotypes.

See below for an example of mucoid vs non mucoid phenotype.

Image:MUCOID.JPG


Assay 2.1 was inspired by: Induction of Capsular Polysaccharide Synthesis by p-Fluorophenylalanine in Escherichia coli Wild Type and Strains with Altered Phenylalanyl Soluble Ribonucleic Acid Synthetase

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