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The strains that we had in possession were ''Lactobacillus delbruckii'' subsp. ''bulgaricus'' (ATCC 11842), ''Lactobacillus delbruckii'' subsp. ''lactis'' (ATCC 10697), and ''Lactobacillus acidophilus''. At first, due to our unfamiliarity, we had a hard time growing up the bacteria. We have written a protocol for growing strains of ''Lactobacillus'' [http://openwetware.org/wiki/Lactobacillus_culture here]. In short, ''Lactobacillus'' requires MRS medium, anaerobic conditions, and a longer incubation time. | The strains that we had in possession were ''Lactobacillus delbruckii'' subsp. ''bulgaricus'' (ATCC 11842), ''Lactobacillus delbruckii'' subsp. ''lactis'' (ATCC 10697), and ''Lactobacillus acidophilus''. At first, due to our unfamiliarity, we had a hard time growing up the bacteria. We have written a protocol for growing strains of ''Lactobacillus'' [http://openwetware.org/wiki/Lactobacillus_culture here]. In short, ''Lactobacillus'' requires MRS medium, anaerobic conditions, and a longer incubation time. | ||
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| + | The major uncertainty in our project was the task of transforming the ''Lactobacillus''. From our research we found a electrotransformation protocol by Serror et al (App and Env Microbiology, 2002). Further research convinced us that electroporation was the strategy we needed to abide by due to the inability to transform ''Lactobacillus'' chemically, given that it is a gram-positive bacteria. | ||
Revision as of 03:35, 30 October 2008
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Binding Assay
Lactobacillus
Our goal was to create a genetically modified Lactobacillus with the ability to secrete the protein p1025. This task would provide the opportunity to contribute the first Lactobacillus parts and devices to the registry, and expand the scope of our iGEM into lactic acid bacteria, which could have numerous applications in health and food technology. This, however, provided many challenges due to our unfamiliarity with this bacteria and a lack of standardized protocols that we enjoy with other laboratory bacteria.
First, we had to design an expression system for the secretion of p1025 in Lactobacillus. This included building a signal sequence and promoter for Lactobacillus bacteria, parts which were not in the registry yet and nobody had any experience with. By fusing these parts with the coding region for p1025 we planned to create a viable expression system.
- The [http://partsregistry.org/Part:BBa_K128004 signal sequence] is a short signal sequence recognized as a secretion tag by several Lactobacillus strains. Our signal sequence was isolated from the 5' end of the prtB gene in Lactobacillus bulgaricus, which codes for a lactose digesting protein that is bound to the surface (Germond JE et al.; Appl Environ Microbiol. 2003). As the attached protein is being secreted, the signal peptide is cut off. Using a cleavage predictor, we extended the protein in order to prevent the cleavage of our p1025 peptide. This part was constructed to use the modified Silver BioBrick prefix and suffix to allow for protein construction.
- The [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128006 promoter] was constructed by isolating the genomic DNA of Lactobacillus and amplifying a native lacS promoter from the genome. The ~200 bp promoter device includes the ribosome binding site, since the part we amplified was from the start of the promoter to the beginning of the coding region. This part uses the standard Biobrick prefix and suffix, as it will never be part of a fusion protein.
The strains that we had in possession were Lactobacillus delbruckii subsp. bulgaricus (ATCC 11842), Lactobacillus delbruckii subsp. lactis (ATCC 10697), and Lactobacillus acidophilus. At first, due to our unfamiliarity, we had a hard time growing up the bacteria. We have written a protocol for growing strains of Lactobacillus [http://openwetware.org/wiki/Lactobacillus_culture here]. In short, Lactobacillus requires MRS medium, anaerobic conditions, and a longer incubation time.
The major uncertainty in our project was the task of transforming the Lactobacillus. From our research we found a electrotransformation protocol by Serror et al (App and Env Microbiology, 2002). Further research convinced us that electroporation was the strategy we needed to abide by due to the inability to transform Lactobacillus chemically, given that it is a gram-positive bacteria.
