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pGLO Transformation

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Heather Delano

on 19 November 2013

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Transcript of pGLO Transformation

pGLO Transformation
Heat Shock
Heat shock is a technique used to make the membrane of the bacteria more permeable. The ranging temperatures create pores and helps the plasmid be taken in.

2 micro test tubes were obtained
1was labeled with +pGLO and 1 with -pGLO
10µl of transformation solution (CaCl2) was added
both tubes were placed on ice
A sterile loop was then used to pick up 2-4 colonies of bacteria (E. coli) from the starter plate provided
The colonies were then dispersed in transformation solution of each tube, using a new loop for each tube
Another sterile loop was then submerged in a tube of pGLO DNA solution and added to the tube labeled +pGLO
Both tubes were then placed on ice for 10 minutes
A real life experiment of this is the pGLO lab.
The pGLO lab is useful in observing the GFP protein in action. In this lab the gene that codes for this GFP protein was stripped away and given to E coli. in an attempt to procure the GFP gene from the Aequorea victoria, or bioluminescent jellyfish, to the E coli sample.

While the tubes were sitting on ice for the 10 minutes, 4 LB agar plates were collected
One plate was labeled with each of the following labels:
LB/amp +pGLO
LB/amp/ara +pGLO
LB/amp –pGLO
The LB is the nutrient broth, amp is the ampicillin, and ara is for arabinose.
Genetic Transformation: In
Layman's terms has the exact meaning: "change cause
by genes".
After the tubes had been on ice for 10 minutes:
The tubes were placed in a foam rack and put in a water bath at 42 degrees Celsius for about 50 seconds.
The tubes were then placed back on ice for another 2 minutes.
The GFP is the protein in the DNA that codes for the Green Florescent Protein found in the bioluminescent jellyfish. This is the protein that was removed from the sample and given to the E. coli in hopes that it would glow.
The genes that make up DNA provide the codes for the making of proteins. The proteins then can insert themselves within the cell and completely change an organisms DNA sequence and even change the physical appearance or phenotype of any organism.
Based on past information and what is known to happen between the E. coli cells and the ampicillin there will be no growth on that plate, one of the controls. The other control with the E. coli, LB, and no ampicillin will have copious amounts of growth. The experimental plates both the positive pGlo with the LB and ampicillin and the pGlo with the LB nutrient, arabinose C, and ampicillin with both have growth although less than the controlled growth without ampicillin. However, the positive pGlo, LB, arabinose C, and ampicillin will also supposedly glow under the ultra violet light.
After the 2 minutes on ice, both tubes were opened and 250µl of nutrient broth was added using new pipette tips for each tube.
The tubes were then allowed to set at room temperature for 10 minutes.
After this recovery time, the tubes were flicked to mix up the bacteria.
10µl of the +pGLO solution was pipetted onto the agar plates labeled LB/amp +pGLO and LB/amp/ara +pGLO
10µl of the -pGLO solution was pitpetted onto the agar plates labeled LB/amp -pGLO and LB -pGLO
The solution was spread out onto the agar plates using a new sterile loop for each plate.
After the solution was spread onto its respective plate, the plates were allowed to set for 1 week until the next lab period.

The plates were then observed in plain light but also under U.V. light.
Data (continued)
Image provided courtesy of a friend named Stephanie.
Our results conclude that the only bacteria to glow contain arabinose, as it initiates the activity of GFP. Only the LB/amp/arabinose plate glowed, as none of the other plates contained any arabinose. This, as well as our two experimental plates seeing bacterial colony growth and our control seeing none, fails to disprove our hypothesis.
A Little Understanding
The gene that codes for GFP is a gene known as arabinose C. The gene is the "C" which utilizes carbon for transcription and translation. This gene is aided in moving from one organism to another by the use of plasmid and calcium chloride. Plasmids contain one or many small circular pieces of DNA that allow adaption within an environment. Within their natural habitat bacteria can transfer plasmids back and forth in a tactful use of survival between many bacteria. Calcium Chloride aids plasmids by allowing the pores to be opened up even more.
Ampicillin is an antibiotic that eradicates E coli on contact. In order to keep that from occurring in transformed cells, sugar arabinose is added to the cells' nutrient medium. These cells appear white on the ampillicin plates containing the arabinose and fluorescent green under UV light. The gene that codes for this resistance to the antibiotic ampicillin is bla. Bla codes for the protein that gives resistance to the E coli colonies.
Fighting Resistance
Each of our plates saw the growth of eighty-three bacterial colonies, while our control, as expected, saw no growth at all.
After calculating, we found our transformation efficiency to be 5.29 x 10^9 transformants per microgram, just below the expected range. This could be the result of adding too much bacterial DNA.
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