09/09/24
Goal: Synthesize Calcium Oxalate crystals and prepare LB medium and Agar-plates for further experiments

Participants: Caitlin

• The exact measurements of Na2Ox and CaCl2 was 0,839g and 0,696 respectively
• The pH of the Tris-HCl buffer was 6,46 - it was made form 0,303g TRIS in 200 mL demineralised water. Then the pH was adjusted using 0,1M HCl and 0,1M NaOH before filling up to 250 mL with demineralised water
• There was a crystal formation immediately after adding the two salts, therefore NaCl was not added to the solution
• It sat for 2 weeks to allow the crystals to fall to the bottom and the water to evaporate

13/08/24
Goal: Make a serial dilution of hydrogen peroxide to test bacterial survival

Participants: Caitlin, Mads, Anna, Viktor, Jonathan
Hydrogen peroxide survivability by use of a growth curve

• The experiment went smoothly apart from having to remove 50 µl of water from the control tube to assure that the concentration of bacteria was constant

17/08/24
Goal: Look at results from the hydrogen peroxide experiment

Participants: Caitlin, (Mads)

• The plates were removed from the incubator and we took pictures before they were disposed of safely

23/09/24
Goal: Transformation of E. Coli

Participants: Caitlin, Mads, Viktor
E-Coli transformation and Oxalate Oxidase production:
We successfully transformed the E. coli and grew it on petri dishes.

• The CaCl*6H2O was supposed to be in a solid form, whereas the water had escaped from the powdered crystal formation, so a rather unknown amount CaCl had been added. The added CaCl consisted of around 0,2-0,3 ml of the solution of the separated crystals. We further added sterile water until the volume was 10 ml to dilute the solution. The original solution with the fully crystalized CaCl is unknown.
• The laboratory did not have any supply of gas left for use with a bunsen-burner, so we resorted to burning ethanol instead to keep an open flame above the petri dishes to minimize risk of contamination.

27/09/24
Goal: Look at results from transformation and transfer to LB-medium

Participants: Caitlin, Marie, Viktor

• The transformation went perfectly.
• All positive controls had growth and all negative controls had none.

30/09/24
Goal: Cell lysis to predate the enzyme tests

Participants: Viktor, Mads

• We successfully isolated the bacteria from the lb medium and used 2 kinds of lysis solution to lyse the cells, so we can later isolate the enzyme.
• 6 of the Eppendorf tubes with cells were lysed with a bio-rad lysis solution that expired in 2017.
• One out of the 6 tubes lysed with 10% Tween 20 solution were lost to the overflow beaker.

1/10/24
Goal: Prove the presence of the enzyme oxalate oxidase.

Participants: Caitlin, Mads, Viktor, (Karoline)

The goal of today's experiment was to prove the presence of the enzyme oxalate oxidase by demonstrating a reduction in oxalate concentration. We carried out the enzyme activity test by measuring the reduction in oxalate concentration over time, with the aim of confirming the presence and activity of the enzyme produced by the transformed E. coli cells.

• During the experiment, we realized that two of our samples might have been contaminated with protease K, which likely affected the enzyme in those samples, preventing them from working as expected.
• Despite this challenge, we achieved the results we were looking for with the remaining samples. The enzyme successfully degraded oxalate, and the measurements fit perfectly with our hypothesis.
• This was the final day in the lab, and no further actions are planned.