Thursday, March 20, 2014

pGlo Lab

At first, we took two micro test tubes and labeled them as containing the plasmid pGLO (+pGLO) and not containing it (-pGLO). We then transferred 250 microlitres of calcium chloride (CaCl2) into the tube. This is used as a transformation solution to help the e. Coli absorb the plasmid and give the desired effects. After placing the tubes on ice, we then transferred some bacteria from the starter plate to the tubes. We then took plasmid DNA containing the pGLO and placed it into the +pGLO tubes. After placing the tubes on ice again for 10 minutes, we placed the tubes in a hot water bath at 42 degrees Celcuis for 50 seconds, then immediately moved them back to the ice for 2 minutes. We then transferred 250 microlitres of LB nutrient broth to the tubes and incubated them for 10 minutes. We then put 100 microlitres of transformation and control suspension onto the plates. We then spread the solution around the plates, closed them up and incubated at 37 degrees celcius until the next day. Then we counted the colonies on each plate and held a uv light to the plate with arabinose to see if it glowed. 

The purpose of the experiment was to genetically alter bacteria. We wanted to see if we could move the genes from the jellyfish plasmids to the e. Coli. The concept we were testing was called genetic transformation. The dependent variables were whether or not we added pGLO and what ager we put the solution on. The relationship was that if there was pGLO and the ager had arabinose and ampicillin then the E. coli glowed and was antibiotic resistant. If there was pGLO but no arabinose then it didn't glow but was still antibiotic resistant. If there wasn't pGLO and there was ampicillin, then all the bacteria died. If there wasn't ampicillin ethn the bacteria svurived. We were really trying to if we could alter the plasmid to meet our needs through genetic transformation. 

Genetic transformation is inserting a gene into an organism to change that organisms traits. In this lab the bacteria E. coli will be altered with genes that code for florecence and for ampicillin resistance. Ampicillin is an antibiotic.  The gene for florecence came from a jellyfish and will cause the bacteria to glow green under the uv light. The "glow" gene is switched on by the presence of a sugar, arabinose, in the ager that the bacteria is put on. The bacteria that was transformed will appear on plates with LB/amp and will glow on plates with arabinose. 

Transformation efficiency is calculated by the total number of cells growing on the agar plate divided by the amount of DNA spread on the plate (in micrograms). The number of cells on the agar plate can be calculated by the amount of colonies, all of which most likely originated one cell. To find the amount of DNA spread on the plate, the number of transformants is divided by the concentration. Our transformation efficiency was 191.251 transformants per microgram. This is much lower than the average 700-800 transform ants per microgram. This lower efficiency was probably caused by a human error. For the -pGLO lb/amp there was no growth, which is what was to be expected as it didn't have the ampicillin resistance. The only plate that glowed was the LB/AMP/ARA because it had the arabinose to fuel the glowing. Any cause for an odd result could've been cross contamination. Even though many precautions were taken against it, like using sterile loops and disposalbe pipettes, but there is a chance that something could've contaminated it while we put it on the table. However, the results that we got do support what we thought the outcome the outcome would've been. Any plate with +pGLO was expected to survive. Any plate that didn't have pGLO could only survive on a plate without ampicillin. 

The lab

We did, in fact, alter the bacteria with the plasmids through genetic transformation. We know that the bacteria was altered because there was growth on the ampicillin plate and it glowed on the plates with arabinose. If the transformation didn't work then all the bacteria on the ampicillin plate would've died and there would've been no glowing bacteria. 
For the +pGLO LB/amp plate, a little growth was seen. This means that some of the cells accepted the plasmid and prevented the ampicillin from killing the bacteria. 

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