A short description of various enzymes useful in recombinant techniques.
Notes on plasmids, and sequencing.
An overview of the polymerase chain reaction.
Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.
General notes Here are a few pieces of information that may be useful to you: You can get petri dishes from Peter Louw; some chemicals as well. You get electroporation cuvettes from Blommie The chloramphenicol is in the walk-in fridge on the second floor. We have our own kanamycin. It is kept at room temp […]
Some transformed cells are fairly fussy in that, unlike other E. coli strains, their plasmids become unstable if the cells are stored in glycerol concentrations greater than 10%. For this reason, the cells should be stored in 8% glycerol and frozen quickly in liquid nitrogen.
The induction of recombinant protein expression is effected by addition of the non-metabolisable lac-inducer IPTG. Although the process is simple in theory, the BL21 cells tend to form inclusion bodies very easily. This reduces recovery dramatically and therefore in the past, each recombinant protein had to be assessed individually for their solubility and expression. This would involve optimising the length of IPTG induction and the temperature, i.e. shorter induction times and lower temperatures are considered best for maintaining recombinant solubility. However, the following two papers have shown that the existing idea that inclusion bodies are dead-end products is incorrect and, with the correct treatment, can be resolubilised, refolded and recovered as active protein. This therefore has the potential to increase recovery and do away with laborious time/temp optimisations for each recombinant protein.
Journal article: Geisbrecht, BV, S Bouyain, and M Pop. 2006. "An optimized system for expression and purification of secreted bacterial proteins." Protein Expression and Purification, 46 (1): 23-32.
Journal article: Cereghino, GPL and JM Cregg. 1999. "Applications of yeast in biotechnology: protein production and genetic analysis." Current Opinion in Biotechnology, 10 (5): 422-427.
Journal article: Carrio, MM and A Villaverde. 2002. "Construction and deconstruction of bacterial inclusion bodies." Journal of Biotechnology, 96 (1): 3-12.
Journal article: Carrio, MM and A Villaverde. 2001. "Protein aggregation as bacterial inclusion bodies is reversible." FEBS Letters, 489 (1): 29-33.
Journal article: Baneyx, F. 1999. "Recombinant protein expression in Escherichia coli." Current Opinion in Biotechnology, 10 (5): 411-421.
For the BL21 cells, there are three potential methods for lysis: Freeze-thaw, Lysozyme digestion, followed by DNAse digestion or mechanical shearing, and Sonication
Transformation is the process of getting the recombinant vector (one containing the gene of interest) from a reaction mixture into cells, such as E. coli BL21(DE3)pLysS cells. To enable the cells to take up circular vector DNA they have to be made competent. The method for preparation of competent cells depends on the transformation method used and transformation efficiency required. For a high transformation efficiency transformation, we use electroporation and electrocompetent cells.
- Protein Amplification and Simple Purification