IGEM:IMPERIAL/2007/Notebook/2007-8-20

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Preparation of Cell Extract

The cell-free protein synthesis reactions will be carried out in a 1.5 mL microtube placed in a water bath set at 37 °C.

  • 57 mM Hepes–KOH (pH 8.2)
  • 1.2 mM ATP
  • 0.85 mM each of CTP, GTP and UTP
  • 2 mM DTT
  • 0.17 mg/mL E. coli total tRNA mixture
  • 0.64 mM cAMP
  • 90 mM potassium glutamate
  • 80 mM ammonium acetate
  • 12 mM magnesium acetate
  • 34 μg/mL l-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid)
  • 1.5 mM each of 20 amino acids
  • 2% PEG (8000)
  • 110 mM phosphoenolpyruvate
  • 15 U/ml pyruvate kinase
  • 6.7 μg/mL DNA
  • 27% (v/v) of cell extract

Total volume for one reaction: 100 micro Litre

Order list

No.ReagentProduct no.QuantityRequiredPrice/ GBP
1ATPA76991g34mg38.60
2CTP30320100mg23mg34.70
3GTPG8877100mg23mg49.90
4UTP94370250mg25mg23.70
5E.coli total tRNA mixtureR4251500UN8.5mg52.60
6cAMP01895100mg11mg13.40
7NH4-acetateA1542250g310mg14.40
8Folinic acidF825925mg1.7mg21.00
9PEG 8000812721Kg1g18.60
  • In vitro experiments using homemade cell extract will start when reagents arrive.

Transformation of Biobricks

  1. 7 biobrick were transformed into XL1-Blue, BL21(DE3) and DH5α
  2. Cells were grown for half an hour and plated
  3. Plates were incubated at 37°C overnight
  • Transformed in XL1-Blue
    • BBa_J23039 [tet-luxI]
    • BBa_I13504 [GFP]
    • BBa_R0062 [pLux (no luxR)]
    • BBa_J37032 [pLux-GFP]
    • BBa_S03119[tet-LuxR]
  • Transformed in BL21(DE3)
    • BBa_R0051 [λcI regualted promoter] (re-clone)
  • Transformed in DH5α
    • BBa_T9002 [Lux-GFP]

Protocols can be found at Electroporation in the general protocols page

Pilot Preparation of Vesicles

Formation of Vesicles

The remaining two of the four suspensions prepared the week before (one well-desiccated and one poorly-desiccated) were used to form vesicles:

  • 2ml of each suspension was used to prepare an interface, according to the protocol.
  • 10x diluted GFP solution was used to prepare the emulsion.
  • Special care was taken to protect the GFP solution from light at all times.

Six samples were prepared:

  • Two following the protocol, with 2ml suspension interface and 100μl of each of two emulsions added:
    • Sample 1: Well desiccated suspension interface with 100μl of emulsion from well desiccated suspension, centrifuge at 120x g for 10mins.
    • Sample 2: Well desiccated suspension interface with 100μl of emulsion from well desiccated suspension, centrifuge at 30x g for 20mins.
  • Four using 2ml of emulsion to form the interface, without further addition of material:
    • Sample 3: 2ml of emulsion poorly desiccated suspension, centrifuge at 30x g for 20mins.
    • Sample 4: 2ml of emulsion from well desiccated suspension, centrifuge at 120x g for 10mins.
    • Sample 5: 2ml of emulsion from well desiccated suspension, centrifuge at 30x g for 20mins.
    • Sample 6: 2ml of emulsion from well desiccated suspension, no centrifuge.

Results

A fluorescence microscope was used to inspect the samples:

  • Sample 1: Sample not looked at.
  • Sample 2: Sample not looked at.
  • Sample 3: Sample not looked at.
  • Sample 4: Tiny, bright, round specks of fluorescence were seen, only under 100x magnification, but they were too small to tell if they were vesicles.
  • Sample 5: Sample not looked at.
  • Sample 6: Tiny, bright, round specks of fluorescence were seen, only under 100x magnification, but they were too small to tell if they were vesicles.

Preparations

No preparations were carried out.

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