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Applications of BBa_I742103
User Reviews
UNIQf601a7a825f326d6-partinfo-00000000-QINU
UNIQf601a7a825f326d6-partinfo-00000001-QINU
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BBa_I742103
derekju -MIT iGEM 2008
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We worked extensively with BBaI742103 and BBaI742123 trying to transform it into Lactobacillus delbruckii subsp. bulgaricus, Lactobacillus delbruckii subsp. lactis, and Lactobacillus acidophilus.
The DNA from the 2008 registry failed to transform and Dr. French, who entered this part, was kind enough to supply us with the plasmids. It turned out the registry wasn't able to transform this plasmid into E. Coli, probably due to non-typical growth conditions.
So this information is on this page, instructions to transform into E. Coli:
- Transform using normal competent E. Coli procedures (electroporation works too, we actually had better results with electrotransforming)
- Select with chloramphenicol at 15 mg/l
- Allow cells to grow for at least TWO DAYS, they take a while to grow and have a low transformation efficiency
- There may high background growth, (probably to the relatively low antibiotic concentration and length of the incubation), make sure to verify that you have miniprepped the actual pTG plasmid. You could send them to sequencing or for a quick check, digest with a few enzymes and run them on a gel. See the plasmid sequence for expected DNA lengths. If you use the biobrick version of the plasmid (BBa_I742123) it has an RFP dropped into it so it's easier to distinguish colonies from background.
Our project aimed to transform Lactobacillus, a lactic acid bacteria, and pTG262 is reported to be able to replicate in Lactobacillus. We tried to electroporate pTG262 into Lactobacillus, and unfortunately we were unsuccessful. According to our research, the most common and successful way to transform Lactobacillus, or similar lactic acid, gram-positive bacteria is via electroporation. We tried various protocols obtained from papers (all of which reported Lactobacillus having a very limited range of plasmids that can be transformed with, due to possible DNA restriction). To view the methods and protocols we used to try to transform Lactobacillus, please visit the MIT 2008 iGEM team wiki. In conclusion, we were unable to transform plasmid pTG262 into Lactobacillus, and are reasonably confident, that pTG262 cannot be electrotransformed into Lactobacillus. For those looking to transform Lactobacillus, other plasmids (such as pJK650 or pLEM415, both of which will be supplied to the registry) should be considered.
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