Scanning Electron Microscopy

A good way to observe the function of a degradation enzyme is to incubate it with the polymer and scan the surface after some time with an electron micrograph. This is a widely used method for studying plastic degradation and we are applying it for characterising Proteinase K enzyme ([http://parts.igem.org/Part:BBa_K1149008 BBa_K1149008]) and observing how it "chews" on a PLA cup.

PLA can spontaneously hydrolyse in water. However, this only happens very slowly and at higher temperatures. Here is an SEM image from the literature (see reference below) where PLA was incubated for 9 and 20 days in deionized water at 58 °C. We are expecting our enzymes to have at least as visible effect in 3 or less days.

method

We cut small pieces of the cup and washed them with ethanol and then with deionised water in order to remove any potential contamination.

The samples 1A, 2A and 3A were incubated with 500uL cell lysate of E.coli MG1655 transformed with BBa_K1149008, for 1, 2 and 3 days.

Sample 1B, 2B and 3B were incubated with proteinase K enzyme solution for 1, 2 and 3 days. (** Units, lets do a LOT)

The negative control A was treated with cell lysate from MG1655 E.coli strain containing the empty vector and the negative control B is a PLA piece with no treatment.

All experiments were carried out at ? degrees at pH ?. We have used a xxxx electron micrograph at the [http://www3.imperial.ac.uk/materials/facilities/em Harvey Flower Microstructural Characterisation Suite], Imperial College.

results

Below come all the fabulous images we shall hopefully have:

references

Effect of NR on the hydrolytic degradation of PLA, Huang et al., 2013