cell lab 10

transformation solution

-It is postulated that the Ca2+ cation of the transformation solution neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells.

heat shock

-The heat shock increases the permeability of the cell membrane to DNA. -The procedure used to increase the bacterial uptake of foreign DNA is called heat shock. -rapid temperature change -must be taken directly from ice, placed into the water bath at 42°C for 50 sec and returned immediately to the ice. The absence of the heat shock will result in a 10-fold decrease in transformants.

how does arabinose work?

-acaC protein is a repressor, binds to promoter for GFP preventing transcription -arabinose binds to araC protein, allowing RNA polymerase to transcribe genes

-Only transformed bacteria that contain the plasmid and express _____ can grow on plates that contain ampicillin. -Only a very small percentage of the cells take up the plasmid DNA and are transformed.

-beta-lactamase -Untransformed cells cannot grow on the ampicillin selection plates.

genetic transformation

-change caused by genes and involved the insertion of a gene or genes into an organism in order to change the trait/s and transfer of plasmids

which plates should be compared to determine if any genetic transformation has occurred?

-dna lb/amp and +dna lb/amp plates b/c cells w/o pGLO won't grow on the amp plates, and cells with it will grow

on which plate would you expect to find bacteria most like the original untransformed e.coli you initially observed?

-dna/lb because the bacteria was removed from the starter plate and didn't have plasmids added

gene codes for a protein that causes the jellyfish to

-fluoresce and glow in the dark -the bacteria will glow a brilliant green color under UV light

name 2 ways to detect if the bacteria has been successfully transformed

1- UV light 2-exposure to ampicillin and survived it

+pGLO, LB/amp: Many transformed colonies of bacteria Colonies appear white. +pGLO LB/amp/ara: Many transformed colonies of bacteria. Colonies appear white when exposed to room light but fluoresce bright green when exposed to UV light. -pGLO, LB/amp: No bacterial growth present on this plate. -pGLO, LB: An even lawn of bacteria is present on this plate. The lawn appears off-white.

A successful experiment will be represented by the presence of colonies on the (+) pGLO LB/amp and (+) pGLO LB/amp/ara plates and the absence of colonies on the (-) pGLO LB/amp plate. Moreover, the colonies on the LB/amp/ara plate should fluoresce green.

how would you change the bacteria's environment to best tell if they were amp resistant?

add amp -if they survive, they are resistant. if not, they aren't resistant

pGLO plasmid encodes the gene for the gfp and a gene for resistance to what antibiotic?

ampicillin

which organism is better suited for total genetic transformation?

an organism composed of a single cell because that one cell can take up the new gene

scientists often want to know if the genetically transformed organism can pass its new traits on to its offspring and future generations. to get this information, which would be better for investigation? an organism in which each new generation develops and reproduced quickly, or one which does this more slowly?

an organism which reproduces quickly, like bacteria

name the 3 genes in the pGLO plasmid

araC, GFP, Bla (beta-lactamase provides resistance to ampicillan/breaks down bacteria)

the gene for the gfp can be switch on in transformed cells by adding what sugar to cells' nutrient medium

arabinose

what 2 factors must be present in bacteria's environment for you to see the green color? why?

arabinose- turns on gfp gene by binding to protein and gene is transcribed UV light- causes gfp to look green

in bio-remediation

bacteria can be genetically transformed w/ genes enabling them to digest oil spills

name one of the genes of the pglo plasmid

beta-lactamase

real-life source of this gene is the...

bioluminescent jellyfish Aequorea victoria

applying biology in the real world by specific manipulation of living organisms, especially at the genetic level, to produce potentially beneficial products

biotechnology

goal of genetic transformation is to...

change an organism's trait/s (phenotype)

pGLO also incorporates a special gene regulation system which can be used to do what?

control expression of fluorescent protein in transformed cells

what is meant by control plate? purpose?

control plate was the -dna plates, so the plates can be compared to see if the bacteria was really transformed. the -dna lb/amp control shows the starter culture doesn't grow on lb/amp

in medicine

diseases caused by defective genes are beginning to be treated by gene therapy by genetically transforming a sick person's cells with healthy copies of the gene involved in the disease

t or f: the GFP gene emits green fluorescent light upon excitation with UV light

false- it's the GFP protein not gene

in agriculture

genes coding for traits such as frost-, pest-, or spoilage-resistance can be genetically transformed into plants

transform bacteria with a gene that codes for a.....

green fluorescent protein (GFP)

genetic transformation means change caused by genes and involves...

insertion of genes AND transfer of plasmids

what is the purpose of arabinose in the experiment of today's lab?

it activated the GFP when put in the culture media

charge on DNA? CaCl2?

negative; positive -CaCl2 allows DNA to cross membrane @42 degrees C -heat shock allows for permeability

can you tell if these bacteria are amp resistant by looking at them on the lb plate?

no, b/c the amp resistant and amp sensitive look similar when cultured

if there are any genetically transformed bacterial cells, which plate/s would they be on?

on lb/amp and lb/amp/ara plates (genetically transformed cells can survive on the ampicillin plates

learn about the process of moving genes from one organism to another with the aid of a ____

plasmid

in addition to one large chromosome, bacteria naturally contain one or more small circular pieces of dna called ____

plasmids

a gene is a segment of dna that provides the instructions for making (coding for) a ____ which gives an organism a particular trait

protein

what traits should the organism have or not have to be sure it will not harm you or the environment?

shouldn't be toxic to humans, plants, or animals should grow well in a lab but not outside lab

plasmid dna usually contains genes for one or more traits that may be beneficial to bacterial ____

survival

what advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

the organism can adapt to conditions and won't have to waste and spend energy producing unnecessary proteins

the recent occurrence of bacterial resistance to antibiotics is due to what?

the transmission of plasmids

process involved 3 main steps intended to introduce the plasmid dna into the e. coli cells and provide an environment for the cells to express their newly acquired genes

to move the plasmid dna - pglo through the cell membrane: 1. use a transformation solution of CaCl2 2. carry out a procedure referred to as heat shock for transformed cells to grow in the presence of ampicillin you must: 3. provide them w/ nutrients and a short incubation period to begin expressing their newly acquired genes

t or f: in nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes

true - allows bacteria to adapt to new environments

genetic transformation involves the insertion of some new dna into the e coli cells using the small circular pieces of dna called plasmids..

which usually contains genes for more than one trait. in this case, the pglo plasmid carries the gene gfp that produced the green fluorescent protein and a gene bla that codes for a protein that gives the bacteria resistance to an antibiotic

transformed cells will appear ___ (wild type phenotype) on plates not containing arabinose, and _____ when arabinose is included in the nutrient agar

white; fluorescent green

did you observe some e.coli growing on the lb plates which did not contain amp/ara

yes, the bacteria w/o plasmids in the plain lb plate

to genetically transform an entire organism...

you must insert the new gene/s into every cell in the organim (bacteria is easiest/best)