Genetic Genetic EngineeringEngineering

What is Genetic Engineering?

• Genetic Engineering = inserting a foreign gene of interest into a host to transcribe and translate a particular protein.

• Ex. Inserting the human insulin gene into bacteria to mass produce it.

Image taken without permission from http://www.medicalprogress.org/uploads/images/insulin%20inject%20WSU%20210.jpg

General Steps

• Obtain the gene of interest (ex. insulin gene)

• Insert the gene into the host (ex. bacteria)

• Allow the host to multiply and express the foreign gene– get your desired protein!– Get lots of cells that can make the protein = clones

The Big Picture

• The inserted gene is transcribed and translated using the RNA Polymerase, ribosomes and other resources in the cell

Plasmids

• Circular DNA

• Extrachromosomal – NOT part of the E. coli genome– “extra” DNA

• Contain a few non-essential genes

• Can give the bacteria additional “traits”– Depends on the genes on the plasmid

• Can be exchanged between bacteria

chromosomal DNA

plasmids

Recombinant plasmids

• Plasmids can be modified in biological labs

• Modified plasmid = Recombinant plasmid

• Plasmids can be used as cloning vectors to get the recombinant plasmid into E. coli– Cloning vectors = way to get the gene of

interest into the host

Transformation

• Process in which foreign DNA is physically inserted into host E. coli cells.

• E. coli that contains recombinant plasmid = Transformed cell Image taken with out permission from

http://summerschool.at/static/irismaria.schoenbrunner/images/transformation.png

Transformation Steps

• Recombinant plasmids and host E. coli are mixed together

• CaCl2 is added

– The Ca2+ ions neutralize the negative charges on plasmid DNA

– Help plasmid enter the membrane

Image taken without permission fromTransformation Animation. Available at http://www.dnai.org/b/index.html

Transformation Steps

• Heat Shock– By rapidly changing the temperature of the

solution, temporary pores are opened in the membrane

– Creates an opening for the plasmids to enter the E. coli

Transformation

• Transformation is not 100% successful

• After transformation– Some cells will contain plasmid = transformed– Some cells won’t contain plasmid = untransformed

• In a later step, you will determine which cells were transformed

E. Coli as a host

• E. coli is a good host because:– Reproduce quickly (once every 20 minutes)– Nonpathogenic (the strain we use is not

harmful)– Genome fully characterized (all genes have

been sequenced)