Within the molecular biology section of our lab, molecular biology techniques are employed to express and purify proteins of interest. This includes construction (e.g. introduction of specific tags) and production of proteins compatible for single molecule experiments such as AFS or for use with magnetic tweezers.
Main equipment include: PCR machine, Electroporator, refrigerated centrifuge, incubators, ultrasonic cell disintegrator - with 3/4 disruptor (sonicator) Western blot, gel electrophoresis, vacuum evaporator, and a fast performance liquid chromatography (FPLC) system.
Bacterial plasmid vectors (bacterial DNA) are used to fuse the protein (or the domain) of interest with specific tags and this vector is grown in E. coli bacteria which are then induced to produce the fusion product. This final product can then used in further single molecule studies.
In our lab all proteins are produced to carry a histidine (his) tag. Ni-NTA beads, which carry high affinity to histidine (Immobilized metal affinity chromatography), can then be used for purification of the protein of interest from the pool of other bacterial proteins. This process is followed by size exclusion chromatography which is based on the separation of proteins accordingly to their molecular weight.
Alongside the his tag, we are able to generate proteins with various additional tags according to the final application. For example; for magnetic tweezers experiments an avidin tag is introduced to the protein of interest. This tag is biotinylated during protein expression and the final biotinylated protein then can be used with avidinated magnetic beads.