The Work in the Lab
After long research and intense discussion, we made our way to the lab in November. We worked towards our highly placed goal for one week long, following in the footsteps of the work of Yoshida and Yamasaki in 1959, of producing a synthetic amylase using two non-canonical amino acids (ethionin und norleucin). Working in this way with the amino acid norleucine was treading a terra incognita. We received support and knowledgable help from two scientists at the Max-Planck-Institute for Biochemestry in Martinsried: Dr. Birgit Wiltschi and Michael Hoesl.
The lab bacterium used, Bacillus subtilis strain 1A55, obtained from the Bacillus Genetic Stock Center, (http://www.bgsc.org/)was supposed to include ethionine and norleucine in its metabolism and finally segregate a synthetics enzyme in its nutrient solution.
The Bacillus subtilis strain we used was a methionine, tryptophan, tyrosine and phenylalanine auxotroph and as such, could not survive outside of its artificially created nutrient medium.
We wanted to use the methionine auxotrophy. We mixed six different nutrient mediums: three based on the standard bacterium medium, called LB, and three based on a different standard medium, called M9.
For LBM, we added methionine as a natural amino acid, for LBN we replaced methionine with
norleucine, and for LBE with ethionine. We produced M9M, M9N and M9E in the same fashion. LBM and M9M (with the natural amino acid bonds) were to be our reference samples to help determine if our Bacillus strain would produce amylases at all: the so-called “reference sample”. The mixture recipes can be found in the lab protocol.
First, the bacteria had to be multiplied to an amount that allowed the planned experiments to be carried out. To do this we mixed pre-culture with a previously prepared medium in a beaker that was then put into an incubator shaker at the bacterium’s ideal temperature of 37°C. The growth was observed through regular photometer measurements. That is how we were able to determine when the bacterium had finished its multiplication. During every check a sample was removed with a pipette and cooled.
The “fully-grown” main culture was centrifuged, the resulting cellular residue removed and filtered. We moved the filtrate with ammonium sulfate in order to cause the omission of the amylase. This process was repeated. We separated the previously used ammonium sulfate from the amylase with the help of a dialysis tube.
The activity of the amylase in the samples that were removed during the week was checked with photometric measurements. The samples with the amylase were mixed with starch and the mixture was incubated at 37°C , allowing the amylase present to break down the starch into sugar. After this incubation time, we added a iodine reagent. The iodine built up in the not yet broken down starch complexes and gave the solution a dark tint. The level of transparency and the corresponding level of breakdown could be measured with the help of a photometer.
A further possible way to measure the amylase activity is with the use of a starch agarose plate. We pipetted the amylase rich sample onto such a plate and the starch contained therein was broken down. This was made visible a few hours later when iodine was added.
Areas in which the amylase had changed the starch to glucose didn’t change color. The more area remained unchanged in color, the more active the enzymes had been.
*Yoshida A. und Yamasaki M.: Studies on the Mechanism of Protein Synthesis into α –Amylase of Bacillus Subtilis, Biochimica et Biophysica Acta 1959, 34, 158-165
*Yoshida A.: Studies on the Mechanism of Protein Synthesis; Incorporation of p-Fluiorophenylalanine into α –Amylase of Bacillus subtilis, Biochimica et Biophysica Acta 1959, 41, 98-103
*Yoshida A.: Studies on the Mechanism of Protein Synthesis: bacterial α–Amylase containing ethionine, Biochimica et Biophysica Acta 1958, 29, 213-214