My last semester as a master student ‘Cellular and Genetic engineering’ has recently started. At this moment of my study, I have had countless lectures about how we scientists can genetically alter micro-organisms to not only study them and try to understand them, but also to implement them in all kinds of sustainable applications. Thus, theoretically I should now be capable of translating any bio-application you can think of into a complex working genetic circuit, eventually resulting in an organism that does exactly what it was intended for. So, bestowed with all this knowledge and a healthy confidence, I threw myself on my master thesis. The research objective of my project is the functional characterization of specific genes in different lactobacilli. These bacteria are the favorite bacteria of the Lebeer lab, living at mucosal surfaces of the human body, including our skin, respiratory tract, gut, urogenital tract etc. Lactobacilli are considered beneficial for normal human functioning and one way to acquire them is via food. The lactobacilli that I am currently investigating were previously isolated form either carrot juice fermentations during the ‘Ferme Pekes’ project or from healthy human vaginas in the ‘Isala’ study. In short, I need to figure out why these specific lactobacilli are so interesting for us and which genes make them so interesting.
Starting the project, I was already picturing all the possible real-life applications that could stem from my research, a new probiotic, an innovative bioactive compound, a cure for cancer or HIV, world peace … who knew? My enthusiasm quickly met the reality of the situation. Lactobacilli aren’t all keen to cooperate with me on this. In fact, they do their utmost best to resist all my efforts to introduce genetic constructs, while this is necessary to elucidate their special traits. So, with a profound reality check, it was time to get back to the basics of genetic engineering!
Genetic engineering altogether comprises an extensive set of techniques to modify DNA either randomly or at very specific sites. One thing that most of these techniques have in common, is that they depend on the introduction of little pieces of DNA along with some molecular cut/paste tools (1.). Once inside, these tiny tools help me to find my genes of interest and specifically alter them by changing their code. For example, by cutting some parts out or replacing them by a self-designed code, the gene becomes disrupted. Because of this, the bacterium cannot access it anymore what causes it to lose a specific characteristic. And so, a mutant is born! By then comparing this mutant with the unaltered bacteria (called ‘wild-type’) in all kinds of tests, I can check which trait my mutant has lost. This way, genetic engineering helps to link genes to bacterial characteristics, enabling us to understand them better. However, most bacteria are naturally not really inclined to take up any additional genetic information from the environment, such as my tools and constructs. This is more scientifically termed as ‘transformation resistant’. Since this is the first critical step to carry out any type of genetic modification, it forms a major bottleneck for their further investigation. Until I can get the constructs inside, I cannot modify them, and I cannot link their traits to their genes. Luckily, scientists have evolved ways to give nature a hand. But, while some bacteria only need a little push, others require complete treatments and still others will stubbornly continue to resist any type of transformation. Unfortunately for my colleagues and me, most lactobacilli belong to these last two categories, what definitely complicates the relationship with our beloved research object.
The most common way to get any type of large molecule into a bacterial cell is by electrotransformation1. In short this means that by giving bacteria a strong electropulse, we can make little holes in their cell walls3. As long as these pores are present, the cells are made permeable and a passage for the uptake of DNA is available2. However, randomly firing some electrical pulses at a bacterial culture will most often not seal the deal. In fact, most bacteria need a thorough preparation by which their cell wall is temporarily weakened and more willingly to become electropermeable3. In this respect, the major challenge for lactobacilli is that these preparation protocols are quite variable between species, and even between strains3. Finding the right electrotranformation protocol is therefore often done via a time-consuming trial and error method. Nonetheless, it remains an extremely important step in successful genetic manipulation. Hence, a large part of my work this year at LAMB consists of the optimization of the electrotransformation protocol of the earlier mentioned fermentative and vaginal lactobacillus strains.
Although this resistance of lactobacilli towards artificial transformation slows down our ability to elucidate their function in the human body and fermented foods, I also must highlight that this does not inherently mean something bad. Whereas it implies that under natural conditions these bacteria are also not prone to take up undesired or dangerous genetic information, such as antibiotic resistance genes or virulence genes. This genetic robustness makes them safer and favors their use in fermented foods and probiotics.
- Börner, R. A., Kandasamy, V., Axelsen, A. M., Nielsen A. T., Bosma, E. (2019) ‘Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech’, FEMS Microbiology Letters, 336(1), doi.org/10.1093/femsle/fny291
- Luft, C. and Ketteler, R. (2015) ‘Electroporation Knows No boundearies: The Use of Electrostrimulation for siRNA Delivery in Cells and Tissues’, Journal of Biomolecular screening, 20(8), pp. 932-942. doi:10.1177/1087057115579638.
- Wang, C., Cui, Y. and Qu, X. (2020) ‘Optimisation of electrotransformation (ETF) conditions in lactic acid bacteria (LAB)’, Journal of Microbiological Methods, 174 (march), p. 105944. doi:10.1016/j.mimet.2020.105944.