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iGEM Freiburg 2022 presents
chAMBER
Standard protocols to cultivate E. coli bacteria.
A complete list of all buffers and stocks, we used for different purposes and how to make them. Recipes for media used to cultivate bacteria in/on are also shown in this register.
Molecular cloning is an essential tool when modifying bacteria for new tasks. As we had many constructs to work with, we did a lot of molecular cloning. We used the following protocols for successful molecular cloning, containing PCR, transformations, DNA clean-ups and -extractions, agarose gels, RE-digest, making competent cells and many more.
As we were not only working on DNA level but also expressing proteins, where we were especially interested in catching mechanisms between proteins, detection of proteins played also a huge role during our project. A main focus here was on SDS-page followed western blots and Native PAGEs.
To detect our compartment-systems and localise them in the cells, we used fluorescence microscopy, guided by following protocols.
The main focus of the project were the BMCs with the full Wiffleball, the flagship of our compartment toolbox. Using the following protocols, we investigated these compartments further.
Since ncAAs were artificially introduced into the bacteria by us, special procedures were required for this. But also their mode of action, e.g. crosslinking via photometric radiation, required special techniques and procedures. The ones we used are listed below.
With the following protocols, Indigo/Indirubin production in E. coli was established and quantified, how much was produced.
These protocols show, how to produce Trehalose in E. coli, measure it with the Trehalose Assay Kit from Megazyme, and quantify, how much Trehalose is in the sample.