MOLECULAR BIOLOGY LABORATORY

Within the molecular biology section of our lab, molecular biology techniques are employed to develop several projects. This laboratory focuses on studying DNA, RNA, proteins, and gene regulation. The tasks performed usually fall into several categories: sample preparation, molecular analysis, genetic manipulation, and data interpretation. Here are the main tasks typically done in our lab:

Sample collection and preparation: Collecting biological samples (cells, tissues, blood, microbes)

  1. Extracting DNA, RNA, or proteins

  2. Purifying nucleic acids

  3. Preparing buffers and reagents

  4. Labeling and storing samples (in ultra-low freezers)

DNA analysis and manipulation:

  1. Polymerase Chain Reaction (PCR) to amplify DNA

  2. Gel Electrophoresis to separate DNA fragments

  3. DNA Sequencing to determine nucleotide order

  4. Molecular Cloning to insert genes into vectors

  5. Gene editing using CRISPR-Cas9

RNA and gene expression studies:

  1. Reverse Transcription PCR (RT-PCR) to study RNA

  2. Quantitative PCR (qPCR) for gene expression levels

  3. RNA isolation and purification

  4. Transcriptome analysis

Protein analysis:

  1. Western Blot to detect specific proteins

  2. ELISA to quantify proteins

  3. Protein purification

  4. Enzyme activity assays

Main equipment include: PCR machines, Electroporator,   refrigerated centrifuges, incubators, ultrasonic cell disintegrator - with 3/4 disruptor (sonicator) Western blot, gel electrophoresis, vacuum evaporator, and a fast performance liquid chromatography (FPLC) system.

PROTEIN SYNTHESIS for single molecule studies: We generate proteins compatible for single molecule experiments such as AFS or for use with magnetic tweezers. 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. 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.