To find out if our Bacillus subtilis culture was truly a catalase (in other words, if the synthetic amino acids had been integrated into its structure) more research steps were required.
The first, relatively simple analysis of the cellular residue and the dissolved proteins contained within that the bacteria produced during its growth, was carried out with the help of an SDS- protein gel. Because of the low material costs, we were able to complete this test ourselves, in our own school lab. The needed acrylamide gel was given to us by the Max-Planck-Institute because, due to its toxic properties, it would not have been safe to do it with a group of students at school.
20 µl of the probe to be examined (collected cellular residue, precipitate and dialysate) were injected with 5 µl of the sample diluent buffer, the liquid was mixed and was put in a centrifuge for a short time to collect it in the tip of a vial. Afterwards there was a 5 minute incubation period at 95°C, during which the strongly lipophile (= fat loving = water hating) part of the sodium dodecyl sulfate (SDS) built up on the surface of the protein sample. As the electrovalence in the watery solution let up, the dodecyl sulfate received a negative charge and the protein joined to it. The solution (with the now negatively charged proteins) was mixed again, centrifuged and placed onto the gel. Then a current of 130 V was put through the sample for 2 hours. The resulting positive charge exerted a pull on the negatively charged proteins. These were - depending on their size - hindered on their way through the gel and correspondingly
separated. As soon as the flow barrier of the lower portion of the gel was reached, the current was turned off, the gel was removed from the carrier plates, dyed and colored and de-colored. On the lamp box, one could easily see the bands of the proteins sorted by size. With the help of the markers on both sides, whose standardized bands had traveled along with through the gel, the mass in kDa (kiladaltons) could be measured. Happily, there was a distinct band at the level of 55 KDa (the size of the catalase we were looking for) in all the analyzed probes.
However, this was not proof that the proteins that were found were catalases and not proteins of a like size. This was very unlikely, but still possible. The SDS had also not shed any light on whether or not the non-canon amino acids norleucine or ethionine were inserted into the catalase.
To find that out a mass spectrometric analysis was needed. Since the required apparatus was quite a bit above our budget (and since the analysis would require a trained expert), the Max-Planck-Institute took over this analysis for us.
To mass-spectrometricly analyze a material, a probe must go through a series of complex procedures: First, the analyte is ionized by various means. This is done by partially breaking it down into its components, out of
which electrons can be removed to achieve a positive charge. The strength of this charge depends on many different factors and determines the uses of the material. Afterwards, the ions are sorted by mass/charge ratio similarly to the process used in chromatography. Then measurements of the split ion packets can be made. These make their way, one after another, across a detector plate that determines their charge and keeps track of any so-called peaks.
In order to evaluate the mass spectrometric results, the charge of each ion must be known. This allows the amount of ions of any particular mass/charge ratio to be determined.
This means that if we know the mass and charge of the synthetic amino acid we are looking for, the mass spectrometric analysis can help to determine if and how often the hoped for manipulated enzyme is present. The exact amino acid chain is obtained and shows on which points the transfer with non-canon amino acids was successful.