Germination Knock-outTo provide an effective mechanism that makes our Nanocillus - the spore expressing a nanobody - safe and ensure that the targeting and drug delivering function is still operative in the colon, we used a germination knock-out, developed by the iGEM team from Munich in 20121.
To understand what the germination knock-out is, we first have to understand the life cycle of Bacillus subtilis. As already mentioned on another page, the endospore is a very resistant form of Bacillus subtilis. This form is dormant and is used to dramatically increase the probability of surviving very adverse conditions such as starvation, heat, UV exposure and extreme pH values2.
Under favourable conditions Bacillus subtilis operates normal cell division. Most of the time too low amounts of nutrition trigger the sporulation process, to create the endospore. In this state the “mother” cell (a single Bacillus subtilis bacterium) creates the endospore within itself by assembling several layers of coat around the bacterial DNA, and releases it the endospore into the environment by lysing itself3.
Under favourable conditions, the endospore starts to germinate. In germination, the endospore goes through a lytic process, which results in the development of a metabolic active and dividing bacterium4.
To now generate a Bacillus subtilis strain that is defective in germination, we had to identify the genes that regulate germination. Fortunately, the iGEM team from Munich already did that and created such a knock-out strain in 2012.
The four genes gerD, cwlJ, sleB and cwlD were knocked-out which resulted in a germination rate lower than 1 in 4.6 * 109 1.
We want to thank the Munich team for sending us this germination knock-out strain.
Coat protein Knock-outsThe germination knock-outs are not the only knock-outs we are using. In our project, we wanted to replace the crust and coat proteins CotZ, CotG, CotB and CgeA with modified versions of these proteins that contain GST or a nanobody as fusion partners. To generate a spore with the maximum amount of fusion protein, we needed strains that cannot produce these coat proteins.
The Bacillus Genetic Stock Center (BGSC) in Columbus Ohio kindly supplied us with these knock-outs and we want to thank them for sending these strains and all the answers they sent us per mail to our endless questions.
1. iGEM team munich 2012, How do sporulation & Germination Work, 2012
2. Driks, A., Bacillus subtilis Spore Coat, 1999, American Society for Microbiology
3. Errington, J. E. F. F. E. R. Y. "Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis." Microbiological reviews 57.1 (1993): 1-33.
4. Paidhungat, Madan, and Peter Setlow. "Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis." Journal of Bacteriology 182.9 (2000): 2513-2519.