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What is the host organism in the transformation lab?
The vector (plasmid) contains the DNA coding for the recombinant ____.
This is the procedure used to increase the bacterial uptake of foreign DNA. This increases the permeability of the cell membrane to DNA.
The liquid and solid media used are called ___. Nutrient broth and nutrient agar. This medium allows E. coli to grow very well.
The plasmid used in this experiment is named ___. It contains the GFP gene and the gene that provides resistance to the antibiotic ampicillin, called beta lactamase.
_________ inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth.
Only _____ bacteria that contain the plasmid and make the beta lactamase can survive on plates which contain ampicillin. Untransformed cell cannot grow on the plates that contain ampicillin.
Transformation solution contains ____ ____ and is pH 6.1. It is believed that the calcium cation in the transformation solution neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells.
The ___ minute incubation period following the addition of LB nutrient broth allows the cells to grow and express the ampicillin resistance protein so the transformed cells survive on the ampicillin selection plates.
____ causes the genes to be transcribed. In the plasmid pGLO, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. When bacteria that have been transformed with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow brilliant green when exposed to UV light.
When arabinose is not in the growth medium, the ___ gene remains turned off and the colonies appear white.
____ _____ literally means change caused by genes, and involves the insertion of a gene into an organism in order to change the organism's trait.
______ plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin.
To genetically transform an entire organism, you must insert the new gene(s) into every cell in the organism. Which organism is better suited for total genetic transformation-one composed of many cells, or one composed of a single cell?
A single-celled organism would be the best recipient for a genetic transformation, because it contains only one cell which needs to take up the new gene.
Based on the above considerations, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning.
A bacterium would be the best host organism. Bacteria are small, single-celled organisms which reproduce quickly and easily.
Describe how you could use two LB nutrient agar plates, some E. coli, and some ampicillin to determine how E. coli cells are affected by ampicillin.
Equal amounts of E. coli cells could be plated on two different LB nutrient agar plates, one which contains just LB nutrient agar and one which contains LB nutrient agar ampicillin. The growth of the E. coli could be compared on the two plates. If ampicillin negatively affects the growth of E. coli, then there should be fewer colonies of bacteria on that plate. If ampicillin has no effect, there should be approximately equal numbers of colonies on both plates.
What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?
Antibiotics usually kill bacteria (are bacteriocidic) or inhibit their growth (bacteriostatic). Thus, there should be few, if any, bacterial colonies present on the ampicillin plate. The presence of any colonies on the ampicillin plate would suggest that those bacteria are resistant to the antibiotic ampicillin.
On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed?
Bacteria which resemble the non-transformed E. coli will be found on the LB/(-) pGLO plate. These bacteria were removed from the starter plate, did not have any plasmid added to them, and were replated on an LB plate. Thus, they are virtually identical to the non transformed starter E.coli.
How much bacterial growth do you see on each plate?
There should be multiple colonies on both LB/amp and LB/amp/ara that received the pGLO plasmid.
There should be no growth on the LB/am(-)pGLO plate.
There should be a lawn of bacteria on the LB(-)pGLO plate.
Many transformed colonies of bacteria (optionally-75) Colonies appear white.
Many transformed colonies of bacteria (optionally -75) appear white when exposed to room light but fluoresce bright green when exposed to UV light.
+pGLO, colonies LB/amp/ara
No bacterial growth present on this plate
An even lawn of bacteria is present on this plate. The lawn appears off-white.
What was the purpose of examining the original pGLO solution with and without UV illumination?
Examination of the original pGLO solution indicates if it glows with UV radiation.
What was the purpose of transferring the DNA+ and DNA- tubes from ice, to hot water, to ice again?
Rapid change in temp makes the cells more permeable to DNA. Process is called heat shock.
Why were the vials incubated for 10 minutes in LB broth rather than transferring their contents directly to the plates?
Ten minutes incubation in lb broth gives transformed cells time to grow and express ampicillin resistance gene. Otherwise, amp media would kill even transformed cells.
What info is provided by lb/-dna plate?
Control to verify e.coli is capable of growing on lb media w/o amp.
What info is provided by the lb/am/-dna plate?
Control that verifies untransformed e. coli cannot grow in presence of amp.
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