Bacterial TransformationRiley Xu

Purpose Insert exogenous DNA into host cells

Modifies genome

Can produce desired traits/proteins

Example: Insulin production

History Discovered by Frederick Griffith (1928)

Non-virulent Streptococcus pneumonia becomes virulent

1944: “transforming principle” found to be genetic

~1970’s: artificially induced competence of E. Coli

Frederick Griffith

Procedure Overview Induce/Buy competent cells

Add DNA

Heat Shock

Incubate

Selective Antibiotic

Competent Cells Some naturally competent cells

Usually during stationary phase

Can induce competence E.g. Adding CaCl2

Shields negative charges on PO43-

Works best with actively growing cells

Make sure to keep cold! Weakens cell surface structure (?)

Heat Shock Add DNA to suspended cells (leave on ice)

Heat shock competent cells to force DNA past membrane Usually at 42oC for 30-90 seconds

Not really sure why it works Forms pores in DNA

Thermal pressure pushes DNA into cell

Return to ice for a couple minutes

DNA Recombination If linear DNA, DNA strand recombines with

chromosomal DNA

Linear DNA transformations don’t work well with induced competence Occurs frequently in natural species though

E.x. Bacillus subtilis

Selective Antibiotics Incubate at 37oC for ~1hr

Transform DNA should contain selective resistance to antibiotic

Time for antibiotic gene expression

Incubate with antibiotics

Other Methods Electroporation

Pulses of several hundred volts per mm

Creates pores in cells

Gene Gun Tungsten or gold coated in DNA

Used mostly for plants

Works ReferencedChin G. c2003-2015. Bacterial Transformation: Definition, Process and Genetic Engineering of E.

coli. Mountain View (CA): Study.com; [accessed 2015 Jun 10]. http://study.com/academy/lesson/bacterial-transformation-definition-process-and-genetic-engineering-of-e-coli.html.

Griffiths AJF, et al. c2000. An Introduction to Genetic Analysis. 7th edition. New York: W.H. Freeman; [accessed 2015 Jun 10]. http://www.ncbi.nlm.nih.gov/books/NBK21993/.

Harris WJ, Barr,GC. 1971 May 27. Mechanism of Transformation in B. subtilis. Molecular and General Genetics. [accessed 2015 Jun 10];113(4):331-344. http://link.springer.com/article/10.1007%2FBF00272333.

Sanford JC, Klein, TM, Wolf ED, Allen N. 1987. Delivery of Substances into Cells and Tissues Using a Particle Bombardment Process. Particulate Science and Technology. [accessed 2015 Jun 12];5(1):27-37. http://www.tandfonline.com/doi/abs/10.1080/02726358708904533

Solomon JM, Grossman AD. 1996 April. Who’s competent and when: regulation of natural genetic competence in bacteria. Trends in Genetics. [accessed 2015 Jun 12];12(4):150-155. http://www.sciencedirect.com/science/article/pii/0168952596100147#.