Molec lab lec 2 - GMO/PCR

Definition of GMO

Organism (plant or animal) whose genetic material
(DNA) has been artificially altered

How are they generally modified

Genetically modified by insertion of foreign genetic
material

Class example of GMO corn

BT Corn (Bacillus thuringiensis insecticidal toxins)

Rice example

Golden Rice (Beta carotene)

Soybean example

Roundup ready Soybeans (Roundup herbicide resistance gene)

Plum example

Virus resistant Plums

Chicken example

Chickens that contain extra gene that prevents avian flu transmission

Mosquito example

Genetically modified male mosquitoes that carry a “self-limiting
gene”

Pros of GMO

Eliminate manual weeding of crops, more successful nutrient utilization by intended crop, enhanced nutrient value of food, and reduced use of pesticides.

How do GMOs help with vaccines

Vaccine delivery, faster development, longer shelf life, and microbial resistance.

Cons of GMOs

• Horizontal gene transfer
• Genes move from intended crop to something else (i.e. crop to weed)
• Changing the organism's effect on the environment
• Effecting viability of offspring
• Some fish have offspring with lower viability
• Allergic reactions to novel proteins
• Reduced Diversity

USDA guidelines for organic food

• an organic farmer can’t plant GMO seeds
• an organic cow can’t eat GMO alfalfa or corn
• an organic soup producer can’t use any GMO ingredients.
• farmers and processors must show
• they aren’t using GMOs
• that they are protecting their products from contact with prohibited
substances from farm to table.

Basic steps of making a GMO

• Identify gene for protein of interest
• Isolate gene
• Engineer gene so that the recipient cell will read it correctly
(continually transcribe it): add promoter, terminator
• Introduce into recipient cells

Method of introducing engineered gene into a cell that includes a bacterium that inserts some of its DNA to a host plant’s genome

Agrobacterium tumefaciens

Method of introducing engineered gene into a cell that includes shooting gold particles coated with engineered DNA into plant cell

gene gun

Method of introducing engineered gene into a cell that includes electric current to create pores in cell membrane

Electroporation

1st step in Agrobacterium tumefaciens mechanism

DNA of gene with desired trait + antibiotic resistance gene with promoter and terminator is added to the Ti (tumorigenic) plasmid inside of the Agrobacterium tumefaciens cell.

2nd step in Agrobacterium tumefaciens mechanism

Agrobacterium tumefaciens cell comes into contact with an undifferentiated plant cell of the plant which leads to the addition of the antibiotic.

1st step of gene gun mechanism

DNA precipitate onto gold particles

2nd step of gene gun mechanism

DNA and gold loaded into bullets and loaded into gun

3rd step of gene gun mechanism

deliver DNA into leaves by shooting

why is gold used for the gene gun

its an inert metal, it will have no effect on the outcome.

1st step in electroporation mechanism

Plasmid DNA is added to electrocompetent cells on ice

2nd step of electroporation mechanism

mix of plasmid and cells are transferred to cuvette in ice

3rd step of electroporation mechanism

cuvette is shocked by electroporator which makes the membrane permeable, and the cells are transformed after they recover in ice

Why are promoters and terminators important for GMOs

Engineered genes must be correctly read and continuously transcribed by the recipient cells

what percent of GMOs us a specific promoter/terminator

Most of the GM plants (~85%) use a specific promoter and/or a specific terminator

Which promoter is mostly used

Cauliflower Mosaic Virus (CaMV) 35S Promoter (constitutively active)

How many base pairs is the PCR product with this promoter

203

Which terminator is most commonly used

Agrobacterium tumefaciens nopaline synthase terminator (NOS)

how many base pairs is the PCR product with this terminator

225