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Team:MIT/Modeling
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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.
Expression System
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 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 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.
Growing the bacteria
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 here. In short, Lactobacillus requires MRS medium, anaerobic conditions, and a longer incubation time.
Transforming the bacteria
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. Finally, we discovered that Lactobacillus strains were only electrocompetent with a very limited range of plasmids, due to possible DNA restriction and other factors.
- plasmid pTG262 - Last year's Edinburgh team worked with the plasmid pTG262 in trying to transform it into different gram-positive bacteria. They had not transformed it into Lactobacillus but were optimistic that it could be, since pTG262 is known to be able to replicate in strains of Lactobacillus. Dr. Chris French was very helpful and graciously supplied us with this plasmid. Working extensively with this plasmid with all of our strains of bacteria, we were unable to successfully transform pTG262 into Lactobacillus, and we have concluded that it is our opinion that pTG262 cannot be electroporated into Lactobacillus delbruckii. We have added characterization to this part's main page and user review page, here.
- plasmid pJK650 and pLEM450 - From the paper by Serror et al., there were two specific plasmids that were able to transform Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis. Dr. Serror from the IRNA in Paris was kind enough to supply us with pLEM415, and Dr. Heinrich from University of Kaiserslautern supplied us with pJK650. These plasmids conferred Erythromycin resistance. Detailed information about pJK650 and pLEM415.
We successfully transformed Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus delbruckii subsp. lactis with a modified version of Serror et al's electrotransformation procedure. Our transformation protocols are found here. We also plan to submit these plasmids, with biobrick sites inserted in them, to the registry.
We are currently working on a method to effectively miniprep plasmid DNA from our Lactobacillus. For information about our protocol for doing so, please click here.
