Supervisor and project title: Jackie Parry, ‘Interkingdom signalling between Saccamoeba limax and Chromobacterium violaceum’
Why I chose this topic
I always found the interaction between molecules very interesting. The mechanisms of disease in the human body, in particular, has always piqued my interest and so this project gave me a way of investigating a topic in line with my interests.
Background to the project
Bacteria communicate with each other using communicating signals known as homoserine lactones (HSLs). When they receive these molecules from each other at a high enough level, they reach something called a ‘quorum threshold’ at this point the bacteria are able to release molecules called virulence factors which help the bacteria to protect themselves from predators, such as amoebas, or launch a synchronised attack against the human body. In this project, the virulence factor produced is violacein.
What was the aim of the project?
The main aims of my project were to discover whether or not the pigment violacein produced by C. violaceum was having an adverse effect on S. limax. This involved carrying out two main methods to determine the toxicity of violacein, then a final experiment to ascertain whether another factor was involved. in order to decide whether violacein was a true contributing factor to the lethality of C. violaceum, three compartments of the bacteria would be studied – HSL, EXT and CAP.
The three compartments were cell-associated product (CAP), homoserine lactones (HSLs) and extracellular product (EXT). Image produced using Biorender.com
How much time was spent in the lab?
I spent around 5 weeks in the lab. It was very different from my usual uni schedule as I could only come in and work every few days. The initial work revolved around the maintenance of my experimental organisms – Chromobacterium violaceum (wild type and mutant) and Saccamoeba limax alongside the ‘prey’ bacteria Escherichia coli DH5α+ and DH5α-. As a result, I would come into the lab three or four times a week, initially on a Monday to subculture my bacteria then again on a Wednesday to re-subculture and use the previously cultured bacteria for metabolic tests. On a Friday I would carry out my feeding experiments and subculture the amoeba S. limax for a repeat experiment on the following Friday. This continued cycle of sub-culturing was crucial for my experiment as if contamination occurred in any of my plates, the experiments would be pushed back an extra week.
What I investigated
These initial experiments involved exposing C. violaceum wild type (capable of producing violacein) and mutant type (incapable of producing violacein) to S. limax and then counting the amoeba growth over several days.
Chromobacterium violaceum wild-type exhibits a dark violet colour which is the violacein.
The Chromobacterium violaceum mutant shown (right) is incapable of producing violacein and therefore is white.
Secondary experiments involved utilising the E. coli DH5α+ and DH5α- to try and disrupt communication between the bacteria and prevent it from producing HSLs and therefore able to produce violacein. The amoeba cells were counted over the subsequent days.
At the same time, I was investigating the metabolic activity of C. violaceum in order to see if it produced any toxic compounds had any antibiotic resistance which could aid in its successful defence against predation.
What I found
It was discovered from analysing the feeding data that only CAP had any effect on the amoebas ability to grow. This could implicate violacein as a lethal product from C. violaceum, however from the metabolic tests it was found that C. violaceum also produces hydrogen cyanide, a compound capable of killing organisms by preventing the production of ATP. However, the HCN was collected from C. violacein in a state where they were provided with complete nutrition whereas the feeding experiments were carried out on agar which provided nutrients for the amoeba. In addition, the HCN was measured qualitatively through remarking the presence of a colour change on a picric acid filter. So to combat these limitations, HCN measurement should be undertaken during the feeding experiments as well as quantified in some way.
Hints and advice
My only advice is to always write everything you do down, no matter how inconsequential it seems at the time when it comes to writing your dissertation you’ll kick yourself for making your life harder and having to search the internet looking for the name of something random, e.g the brand name of the antibiotic discs you use (guilty). Also, don’t stress out too much about the outcome of your work, the results don’t matter nearly as much as how you interpret them- so always make sure you understand your work! Finally, when it comes to writing your lit review don’t be afraid to critique other scientists- if you don’t think their paper is particularly good or helpful- say so! But make sure you give reasons for this!
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