Team:Bielefeld-Germany/Protocols

From 2012.igem.org

Protocols

Molecular

In this section of our protocol pages you can read more about our methods for cloning and BioBrick assembly.

Genetic engineering is a basic tool of synthetic biology. With the help of standardized DNA building blocks (BioBricks) it is fairly easy to create new and modify existing natural systems. The methods we have used in our project to create BioBricks and to modify, mutate, transform and analyse DNA are presented in this section. Methods used: Electroporation; chemical transformation; Standard, Freiburg, Gibson and 3A BioBrick assembly; restriction analysis; colony PCR; site directed mutagenesis.

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Production

These are the protocols for the cultivations and the downstream processing.

Before one is able to work with a cell-free system based on biological material, the needed proteins have to be produced and purified first. These methods and the ones we used to characterize our generated BioBricks in vivo are presented in this section. Used methods: Cultivations in shaking flasks and in differnt bioreactor systems with a working volume up to 6L; mechanical lysis of cells, protein clean-up from lysed cells, Ni-NTA- and TALON-columns and chromatography.

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Analytics

Protocols for the analytical methods we used.

DNA and proteins are very small and cannot be seen by the naked eye. To control the success and the results of your upstream and downstream processes, analytical methods are necessary that give reliable results to make DNA or proteins in any way visible for you. The analytical methods we used in our project can be found in this section. Used methods: Fluorescence measurement; SDS-PAGE; MALDI-TOF; HPLC; LC-ESI-qTOF-MS/MS; molecular beacons; extraction.

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Immobilization

Protocols for the immobilization

Immobilization offers the opportunity to develop a degradation system. Therefor we tried to find a method to immobilize the produced lacasses. For the immobilization we tried tow different methods. First silica beads, which were already available in our lab, because they were used the year before. And later CPC-(controlled pore carrier)-beads.

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Material

Chemicals, enzymes and kits we used in our lab work.

Chemical and biological reactions need defined conditions to work as expected. The chemicals, enzymes, kits, buffers and media we used in our project are listed in this section.

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