Team:UPF-CRG Barcelona/Experiments



Experiments - Polybiome

Experiments

Functionalisation Experiments

Spectrophotometry assay
Auxotrophy of PACO strain
The aim of this test was to assess the behaviour of the auxotroph strain PACO at different levels of polyamines being 10mM of Putrescine and 5 mM of spermidine and 0.0195 mM of Putrescine and 0.00975mM of spermidine the highest and the lowest concentration of polyamines respectively. As a control DH5alfa strain was used. The schema of the 96 wells plate can be observed below.

Grillo 1

Procedure
  1. Both strains, DH5α and PACO, were harvested in a liquid culture with optimal conditions until saturation.
  2. Optical density of both cultures was mesaured and the concentration was readjusted to be 0.1 A.
  3. Growth mediums were cleaned twice in order to ensure a perfect control over the growing conditions during the test.

  4. A M9-polyamines Mix (x2) was prepared.

    M9-polyamines Mix (x2)
    22.12 μL Putrescine (1000x)
    22.12 μL Spermidine (1000x)
    2.5 mL
  5. Last column of the 96 wells plate was filled with 100μL of M9-polyamines Mix (x2) and 100 μL of M9.
  6. 100μL of M9 was placed at each well except for the last column.
  7. 100μL of M9-polyamines Mix (x2) was placed at well A-11 and resuspended.
  8. 100μL were transferred from well A-11 to well A-10.
  9. 1:2 serial dilutions were performed all along the row until well A-1, discarding the 100μL remaining in the last step.
  10. This procedure was repeated for all the remaining rows of the 98 wells plate.
  11. Finally, 100μL of cells with the convenient antibiotic were placed at each well; form A-D with DH5α strain and from E-H with PACO strain.

Charaterization of BBa_K2118004 device

The aim of this test was to assess the behaviour of the auxotroph strain E.coli MA255 (Yale) with an without the device BBa_K2118004 at different levels of polyamines being 2mM of Putrescine and 2mM of spermidine and 2μM of Putrescine and 1μM of spermidine the highest and the lowest concentration of polyamines respectively. As a control DH5alfa strain was used. The schema of the 96 wells plate can be observed below.

Grillo 2

Procedure
  1. Both strains, DH5α and Yale with and without device, were harvested in a liquid culture with optimal conditions until saturation.
  2. Optical density of both cultures was mesaured and the concentration was readjusted to be 0.1 A.
  3. Growth mediums were cleaned twice in order to ensure a perfect control over the growing conditions during the test.

  4. A M9-polyamines Mix (x2) was prepared.

    M9-polyamines Mix (x2)
    22.12 μL Putrescine (1000x)
    22.12 μL Spermidine (1000x)
    2.5 mL
  5. Last column of the 96 wells plate was filled with 100μL of M9-polyamines Mix (x2) and 100 μL of M9 minimal media (with aminoacids and salts). [link here]
  6. 100μL of M9 minimal media (with aminoacids and salts) was placed at each well except for the last column.
  7. 100μL of M9-polyamines Mix (x2) was placed at well A-11 and resuspended.
  8. 100μL were transferred from well A-11 to well A-10.
  9. 1:2 serial dilutions were performed all along the row until well A-1, discarding the 100μL remaining in the last step.
  10. This procedure was repeated for all the remaining rows of the 98 wells plate.
  11. Finally, 100μL of cells with the convenient antibiotic were placed at each well; form A-C with Yale with device, D-F with Yale strain and G-H with DH5α.

Characterization of absorbance peak of polyamines

The aim of this characterization was to be able to determine the amount of polyamines in a medium just by using an spectrophotometry assay. Several concentrations of Putrescine were used in the assay being 10mM and 0.016mM the highest and the lowest concentration respectively. The schema of the dilustions can be observed below.

Polyamines experiment

Procedure
  1. Polyamines mix at maximum concentration was prepared. Polyamines Mix (10mM) | ———— | 17.7 μL Putrescine (1000x) | 1.982 mL H~2~O |
  2. 1mL was placed in a fused quartz cuvette.
  3. A wavelength sweep measurement of abosrbance was done from 1100nm to 190nm.
  4. 0.6mL were discarted from the Polyamines Mix (10mM) and refiled with 1.6mL of H~2~O.
  5. 1mL from Polyamine Mix (2mM) was used for next measurement.
  6. Several 1:5 dilutions were performed until reaching a Putrescine consentration of 0.016mM.