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Revision as of 22:27, 24 October 2007

System Parts

educatETH E.coli consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our In the Lab page!). Four of them (4,5 and 8,9) form together two functional system units, but they have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids. Are you interested in the structure, mode of action or purpose of individual parts? Just click on the specific links in the third column. This will directly guide you to the entries in the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. All 11 educatETH E.coli parts are listed in the following table:

Those 11 basic parts have been further assembled into intermediates and composites, which in turn enable the system to operate:

Many of the above mentioned parts contain double promoters. This promoter constructs contain two independent classes of operator sites and can therefore be regulated by two different types of molecules. Double promoters form the basis of the multi-inducible toggle switch and hence the memory of our learning system. This concept could also help future projects in developing devices and systems that need extended regulation. In the following, we introduce a selection of first generation double promoters:

In order to test if the concept of the proposed double promoters is working, simple proof of concept (PoC) parts have been constructed. The PoC promoter, which shows strong similarites to [http://partsregistry.org/Part:BBa_I739102 BBa_I739102], consists of two TetR operator sequences linked to a constitutive promoter. In contrast to the other double promoters , the PoC promoter is only single regulated. The PoC intermediate is part of the PoC composite the concept can be tested with, that is, EYFP production. The p22cII coding region is included to ensure the functionality in multicistronic constructs.

Although the parts have been synthesized by GENEART and also were shipped in their high-copy plasmids, we desided to change the cloning vectors. This strategy is based on the fact that fluorescent proteins are potentially harmful to the cells. The parts containing DNA sequences coding for these reporter proteins are therefore supposed to be cloned in low-copy number plasmids. In contrast, the constitutive subsystem parts can be cloned into a well characterized and working high-copy plasmid. Summarized, the following plasmids have been used:

Two E. coli strains have been used in this project. In the beginning, only the Top10 strain was worked with. But in a later stage of the project, using JM101 cells seemed to be more productive since they are growing faster than the Top10 cells.