pGLO Lab

PURPOSE 

   The purpose of this lab was to help us understand genetic transformation because moving genes from one organism to another through help of a plasmid. We did genetic transformation in order to determine the rate of a plasmid carrying a gene that transforms into E. Coli. 

INTRODUCTION

Genes are changed by genetic transformation because a certain gene is placed into an organism. Antibiotic ampicillin treats certain bacterial, but pGLO is resistant to it. When sugar arabinose is in the cell it allows GFP to be activated in transformed cells. We hypothesized that the bacteria will gloom the tray with the ampicillin and they will not glow on the one without arabinose. Arabinose will turn on the glowing expression. 

METHOD

1) First things first, we grabbed two micro test tubes, labeled one +pGLO and the other -pGLO, and then four LB nutrient agar plates and labeled them as shown below.

2) Using a sterile transfer pipet, we dropped 250µL of transformation fluid (CaCl2) into each tube and then put them on ice.

3) Using a sterile loop, we took one colony of E-coli from a starter plate and put it into the +pGLO tube. We then did the same for -pGLO but using a new loop.

4) Using yet another new loop, we transfered pGLO plasmid DNA into the +pGLO tube.

5)The two tubes were then kept on ice for ten minutes, followed by a 50sec heat shock delivered in a water bath set to 42 degrees Celsius, followed by yet another ice incubating period for two minutes.

Ice incubation

Heat shock

6) Finally, we added 250µL of broth to each test tube and then using a pipet we pipetted 100µL of the test tubes’ contents onto the appropriate nutrient agar plates.

 

All packed up until the next day!

DATA

Aaaaaand our experiment failed. After being exposed to ampicillin, all of our E-coli died. Luckily for us, we were able to get our hands on the another groups last two agar plates to show what the results should have been. 

DISCUSSION

   The results of the two control plates were as expected. The untransformed E. coli prospered in the agar without ampicillin, covering the surface of the plate almost entirely, and was eradicated from the agar with ampicillin. Both of the experimental plates contained ampicillin, which was meant to select against untransformed E. coli and leave a few colonies of E. coli that had been transformed by pGLO, while the fourth and final plate also included arabinose to make the transformed E. coli a fluorescent green under UV light. The expected result would have been several isolated colonies of nonfluorescent transformed E. coli on the first experimental plate and a similar number of colonies of fluorescent, ampicillin-resistant E. coli on the second, as observed in the agar plates from the other group's lab. The other group was found to have 30 colonies of transformed fluorescent E. coli, which was calculated to be a transformation efficiency of 191.25 transformants per microgram of pGLO DNA spread on the plate. However, as all E. coli was eradicated from both plates in this experiment, it can be concluded that the transformation failed. The process produced zero colonies and a transformation efficiency of zero. The gene for ampicillin resistance from the pGLO plasmid was not incorporated into the E. coli's DNA, so the E. coli on the agar plates containing ampicillin was killed.

     Errors may have occurred at many steps along the way. Both control plates resulted as expected, so the error was probably not in the E. coli or the agar plates. Also, several other groups used the same pGLO and had successful transformations, so the chances of having faulty pGLO are also low. Thus the most likely option is that the E. coli underwent too much heat shock, allowing its proteins to denature irrevocably, or too little heat shock, preventing the pGLO genes from bypassing the cell membranes. The problem may also have occurred immediately following the heat shock if the tubes had not been in close enough contact if the ice to allow the cell membranes to reform. As other groups were able to transform bacteria with a higher efficiency using the same materials and procedure, to improve this experiment would be a simple matter of following the procedure and the timing of the steps more precisely.

CONCLUSION

   The experiment was ultimately unsuccessful in transforming E. coli into a fluorescent, ampicillin-resistant bacteria. The transformation efficiency was zero.