Exam 2 T 10/30 at 7:30-9pm Review Th 10/25 at 5-7pm in WRW 102 and/or in class Bonus #1 due 10/25 in class

Genetic Engineering: Direct manipulation of DNA

Bacteria can be modified or serve as intermediates

a typical bacteria Bacterial DNA plasmid DNA

A typical bacterial plasmid used for genetic engineering

Moving a gene into bacteria via a plasmid

Bacterial DNA plasmid DNA What problems exist for expressing eukaryotic gene in bacteria?

Reverse transcriptase can be used to obtain coding regions without introns.

Fig After RT, PCR will amplify the gene or DNA

Moving a gene into bacteria via a plasmid RT and PCR

Restriction Enzymes cut DNA at specific sequences

Restriction enzymes cut DNA at a specific sequence

Fig 20.2 Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends

A typical bacterial plasmid used for genetic engineering

Using sticky ends to add DNA to a bacterial plasmid Fig 20.2

If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself.

Fig 20.2 The plasmid is treated with phosphatase to remove the 5’-P, preventing self- ligation

Fig 20.8 Transformation of bacteria can happen via several different methods.

Fig 7.2 Bacteria can take up DNA from the environment

Fig 20.8 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Electroporation Heat shock Osmotic Stress

How can you know which bacteria have been transformed, and whether they have the insert?

Figure 20-5 Fig 20.5 Resistance genes allow bacteria with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic

Figure 20-5 Fig 20.5 Is the insert present? Plasmids with the MCS in the lacZ gene can be used for blue/white screening…

A typical bacterial plasmid used for genetic engineering

Figure 20-5 Fig 20.5 Intact lacZ makes a blue color when expressed and provided X-galactose

Figure 20-5 Fig 20.5 When the lacZ gene is disrupted, the bacteria appear white

Blue/white screening: Transformed bacteria plated on antibiotic and X- gal plates. Each colony represents millions of clones of one transformed cell.

Successful transformation will grow a colony of genetically modified bacteria Fig 20.4

Inserting a gene into a bacterial plasmid RT and/or PCR

Millions of Hectares Texas = 70 ha Bacteria can be used to transform plants Global area planted with GM crops

Agrobacterium infect plants, inserting their plasmid DNA into the plants genome.

Fig Agrobacterium infect plants, inserting their plasmid DNA into the plants genome.

Fig By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant.

Fig The generation of a transgenic plant Grown on herbicide

How do you know whether the gene you want to express has the correct sequence?

DNA sequencing

The structure of 2 ’,3 ’ -dideoxynucleotides Fig 20.15

Fig The dideoxy sequencing method

Fig The dideoxy sequencing method

Fig Gel produced by the dideoxy sequencing method

Fig Computerized sequencers use a similar method

Creation of Caenorhabditis elegans transgenes Figure 20-28

Creation of Drosophila melanogaster transgenes using a transposon Fig 20.29

Creation of Mus musculus transgenes Figure Fig 20.30

…now enjoy making Frakencritters.

Exam 2 T 10/30 at 7:30-9pm Review Th 10/25 at 5-7pm in WRW 102 and/or in class Bonus #1 due 10/25 in class