Calcium chloride heat shock transformation
Calcium chloride heat shock is a common method of transformation used with E. coli cells.
It is not precisely known what the mechanisms are but the current theory is that the DNA enters the cell through pores in the cell membrane known as adhesion zones. Rapidly growing E. coli cells contain a large number of these adhesion zones where the inner and outer cell membrane fuse with each other. The hydrophilic phosphate groups on the surface of the lipid bilayer are strongly negatively charged as is the DNA so normally they repel each other. A solution of calcium chloride (CaCl2) is added to the cells and the calcium makes the environment more electrostatically neutral so the DNA can be closely associated with the membrane. The cells which have been kept at a cool temperature the entire time are then "heat shocked" by rapidly transferring them from approximately 0 C to 42 C. This creates local strong temperature gradients and disruptive currents in the system that results in some of the DNA being taken up into the cell through the adhesion zone pores.
Notes
This method was first discovered to increase the rate of bacteriophage infections by the uptake of viral DNA by the cells.[1]
Inoue et al. 1990 published a method to store cells in a competent state (able to take up DNA during transformation) that can greatly increase efficiency when a large number of transformations are being done over time.[2]
Li et al. 2007 constructed a MEMS (Micro Electro Mechanical Systems) device that greatly increases transformation efficiency with much smaller volumes.[3][4]
Panja at al. 2008 found that the heat-shock (and a following cold-shock) step released lipids and proteins from the cell membrane and facilitated pore formation that might contribute to the uptake of DNA. Furthermore, repeated cycles of heat and cold shock, up to the third pair, increased transformation efficiency.[5]
Sarkar et al. 2002 found that ethanol removed lipopolysaccharide (LPS, lipid-phosphate membrane molecule with a cell surface O-antigen polysaccharide covalently attached) and lowered transformation efficiencies, suggesting DNA association with LPS molecules may be a key part of calcium chloride heat shock transformation.[6]
References
- ↑ Mandel, M., & Higa, A. (1970). Calcium-dependent bacteriophage DNA infection. Journal of molecular biology, 53(1), 159-162. [1]
- ↑ Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23-28.[2]
- ↑ Li, S., Anderson, L. M., Lin, L., & Yang, H. (2007, January). DNA transformation by local heat shock. In Micro Electro Mechanical Systems, 2007. MEMS. IEEE 20th International Conference on (pp. 533-536). IEEE.[3]
- ↑ Li, S., Anderson, L. M., Yang, J. M., Lin, L., & Yang, H. (2007). DNA transformation via local heat shock. Applied physics letters, 91(1), 013902.[4]
- ↑ Panja, S., Aich, P., Jana, B., & Basu, T. (2008). How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?. Molecular membrane biology, 25(5), 411-422.[5]
- ↑ Sarkar, S., Chaudhuri, S., & Basu, T. (2002). Mechanism of artificial transformation of E. coli with plasmid DNA-Clues from the influence of ethanol. CURRENT SCIENCE-BANGALORE-, 83(11), 1376-1379.[6]
