The following are quick descriptions of our experiments, why we made them and their results. To see our more precise lab notes and protocols, please see the Lab Notebook tap above.
The GMO Experiment - Transformation and Enzyme Test
We wanted to prove that it was possible to transform bacteria with our plasmids, and produce the enzymes that were able to break down oxalate.
To prove it we wanted to create four different agar plates that would contain the following; two with antimicrobial peptides and two without, with one being with the plasmid insert at 2 without.
Our transformation was done with the method of heat shock and we utilized a nickel column to collect the protein of interest. And the enzymes were activated with the use of L-arabinose, where we measured the amount they were able to break down using a titration with potassium permanganate and sulfuric acid
E.coli Resistance to Hydrogen Peroxide
To make sure we wouldn't end up with zero results, we wanted to test how well E. coli would be able to produce enough Catalase, a hydrogen peroxide degrading enzyme. This was important since the breakdown of our enzymes releases hydrogen peroxide. We knew that bacteria have pathways to break down hydrogen peroxide but we wanted to test how well this feature was, to make sure it was safe. The experiment would hopefully show us whether we needed to add the gene that codes Catalase to our plasmid or if the bacteria were naturally able to survive the increased concentration.
This was simply done by making LB medium containing different levels of hydrogen peroxide, and a control without to determine the survival rate at different levels.
Results
The results showed that when the concentration of hydrogen peroxide was 0,01% or lower there was no significant decrease in bacterial growth compared to the control. Though at 1% hydrogen peroxide the bacterial growth was severely stunted compared to the control (the solution with bacteria bubbled when the hydrogen peroxide was added showing its oxidative capabilities.) as well as that the 0,1% also had significantly less growth. This shows that concentrations from about 0,1% and above are harmful for the bacteria and therefore most likely also for our cells.
From the experiment we can conclude that at low concentrations of hydrogen peroxide the bacteria are not harmed. Though to go further with the experiment we should test what the concentrations of oxalate are in the patients so that we have an estimate of how much hydrogen peroxide is produced.
