Intro: Genes can be spliced into a cell in order to alter the makeup of the cell. In our lab we spliced a "pglo" gene into EColi bacteria in order to make it resistant to ampicillin. This the presence of the pglo gene will allow the EColi to glow under an Ultra violet light. Without the pglo the bacteria will die once introduced with ampicillin. The activator for the pglo gene is arabonose sugar, so when arabonose is present the bacteria will grow more.
Methods: First we transfered 250 ul of CaCl2 into test tubes labeled +pGLO and -pGLO and then placed them in ice.
Then pGLO plasmid DNA was added to the +pGLO tube but no the -pGLO tube. The test tubes were placed in ice for 10 minutes before being placed in a hot water bath for 50 seconds to heat shock them.
The tubes were placed back in ice for 2 minutes and then removed. Then 250 ul of LB nutrient broth was added to both tubes and they were mixed.
Finally 100 ul of the tubes solution were added to their respective plates, +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB.
The suspension was spread evenly on the plates and then they were left alone for a day.
This is the picture of the base bacteria that we took the sample from to spread it to all of our plates
(from left to right top to bottom) ampicillin was added to the plate but the ere was pglo so it was able to resist and survive, thus showing some growth. then there is lots of growth on the LB because there was just regular conditions for the bacteria to grow in. next there was marginally more growth in the amp/ara because there was the activator arabinose which makes the pglo more active. and because of the pglo the ampicillen did not kill the bacteria. finally there is nothing in the last tray because the bacteria did not have the pglo gene in order to make it resistant to ampicillin so it died.
Discussion: In our base group with just the EColi gene there was massive amounts of growth, and the entire dish was full. Where in the plain EColi gene introduced with ampicillin there was nothing in the tray because all the bacteria was killed. Then in the pglo enhanced EColi gene there was actually growth on the ampicillin tray, but there was not that much. This is because not all of the bacteria cells become resistant to the ampicillin. So the ones that weren't resistant died. Then finally the pglo bacteria introduced to the ampicillin and arabonose sugar was able to grow more because pglo becomes active when arabonose is present so it grew more than without the arabonose. But not nearly as much as the base dish.
Conclusion: The objective and process of this lab was to move genes from one organism to another by using the help of a plasmid. The pGlo plasmid we used had a gene which was encoded for GFP (green fluorescent protein), was altered with both arabinose or ampicillin. Each of the four dishes in which the plasmid was placed had a different component as explained above. The dish labeled with LB/ARA/AMP was able to glow in the dark which gave us a clear result on the test. The others did not, as predicted.
