BIO 206L- Exercise 10: Transgenic Organisms: Bacteria and Nematodes

Why are C. elegans great model organisms?

-Complete cell fate mapping -easy to manipulate genes -simple internal structures, easy to view -many generations over short period of time

Gene editing

-microinjecting -gene bombardment -crispr cas-9

What are the steps of a bacterial transformation and the purpose of each?

-obtain chemically competent E. coli -pipet E. coli into a chilled tube -pipet pLAF plasmid DNA into tube -mix by flicking/tapping -incubate on ice for 15 minutes -heat shock for 30 seconds -ice for 5 minutes -add LB Broth -incubate in 37 degree Celsius -incubator for 40-60 minutes

2) What is the size of an adult C. elegans?

20-25 micrometers

Chemically competent cells

Can take up foreign DNA due to treatment of high concentration of CaCl2 or other divalent cations. only a tiny percentage of competent cells will become transformed and will successfully incorporate a plasmid.

4) True or False (correct if false): All of the bacteria growing on LB only plates are transformed.

False, None are transformed

3) True or False (correct if false): You can see the synaptic vesicles in the transgenic C. elegans using brightfield microscopy.

False, only fluorescence microscopy

Where are the synaptic vesicles found in nematode? What is their purpose?

Found all throughout the nematode, they are labeled with GFP marker *synaptic vesicles transport neurotransmitters to synaptic cleft, where neurotransmitters are released.

Are plasmids essential to bacterial life?

No, they allow for bacterial sex, or have genes that confer resistance to an antibiotic, but they are not essential to its life. can provide a selective advantage to cells that possess them.

Which bacteria will grow on media containing ampicillin?

Only bacteria that has been transformed with pGLO plasmid will survive and grow on media containing ampicillin

Arabinose operon

Only when arabinose (sugar) is present in the growth media is the GFP gene transcribed (genotype). When the GFP gene is expressed, the transformed bacterial cells will glow under UV light. (phenotype) Environmental conditions: the presence of arabinose in growth media.

Vector

Recombinant plasmid A researcher may insert a useful animal gene into the plasmid and then insert that recombinant plasmid (vector) into a bacterial cell. Plasmid is then replicated in the host cell, which results in synthesis of many copies of useful animal gene. ex: a plasmid with an ORI, Promoter, restriction sites, and genes that code for lactose digestion (lacZ)

How is bacteria exposed to ampicillin?

Scientist exposes chemically competent bacterial cells to plasmids that encode a gene for resistance to antibiotics. Then spreads the exposed bacteria uniformly over a plate containing ampicillin. Only the transformed bacteria will survive and divide on the plate with ampicillin!!! -e.coli cell can have 1016 clones in less than 18 hours

Plasmid

Small, circular DNA molecule that is distinct from, and replicated independently of the bacterium's chromosomal DNA.

Why are bacterial plates grown inverted in 37 degree incubator overnight?

So the condensation doesnt drip on the bacteria

5) True or False (correct if false): Bacteria use plasmid to for sex as well as other uses such as killing other bacteria.

True

1) Why is it important to NOT vortex the chemically competent E. coli cells?

bc they are fragile

the pGLO plasmid

engineered plasmid used as a vector for creating genetically modified organisms. contains: 1) ori (origin of replication) 2) ampicillin resistance gene (bla) 3) gene that codes for green fluorescent protein (GFP) 4) multiple cloning site (MCS) 5) araC gene

What happens if the bacteria are not thawed "gently" or not kept on ice? What happens if the bacteria are at 42 degrees Celsius for longer periods? (heat shock)

enzymes that replicate the DNA can become denatured

araC

in absence of arabinose, araC protein prevents transcription of GFP gene, opposite in presence of arabinose.

Transgenic organism

made from a gene from the DNA of one species being inserted into the DNA of another species in this exp: E. Coli will be genetically modified to express a gene isolated from the bioluminescent jellyfish. The gene codes for green, fluorescent protein (GFP).

C. Elegans

transparent nematodes transgenic GFP protein fused to synaptic vesicle proteins expressed in C. elegans' neurons. Using a fluorescence microscope, will be able to see location of synaptic vesicles in a living nemotodes nervous system