PGLO Transformation Lab

Purpose

     In this experiment, we were observing bacterial growth and transforming the bacteria. The process allowed us to further understand how transformation occurs, what happens biologically to the bacteria when genes are moved, and the significance this process has on life in both prokaryotic and eukaryotic cells.

Intro

     Transformation is a process used in prokaryotic as well as eukaryotic life cycles to genetically modify a cell by introducing  separate DNA into the cell with the help of a plasmid. Plasmids are genetic structures in a cell that are often used in laboratories to manipulate genes. They are small, circular DNA molecules in the cytoplasm of cells that carry supplementary genes and replicate independently of the chromosome. They can even lead a cell to being antibiotic resistant. The pGLO plasmid contains genes for green florescence (GFP) as well as a gene that has resistance to the antibiotic ampicillin. Scientists such as biochemists sometimes use a method called heat shock which is where cells are put into a higher than ideal temperature of the organism. This method opens up pores in the plasma membrane due to the sudden increase in temperature, allowing plasmids to enter the bacterial cell.

Methods

Because this lab involved E.coli and making it antibiotic resistant, extra precautions, sterile equipment, and good lab techniques were used while doing this lab. No one was harmed during this genetic transformation.

Micro test tubes were labeled +pGLO and -pGLO and transformation solution was added into each tube.

Using sterile loops, we picked up the E. Coli and placed them into both test tubes.

All the while both test tubes were immersed in a cup of ice in order to keep them cold. Then we grabbed another sterile loop and obtained pGLO plasmid DNA where we only placed into the +pGLO and not the -pGLO. After ten minutes sitting in the cup of ice, we heat shocked the test tubes. This meant putting both tubes into a 42 degree Celsius water bath for 50 seconds then placing it back into the ice immediately afterwards. Next, LB nutrient broth was placed into both test tubes. Finally, we placed the +pGLO into the LB/amp and LB/amp/ara transformation plates, the -pGLO was placed into the LB/amp and LB control plates, and gently spread the the liquid across the plate. The only thing left to do was to put them into the incubator and wait.

Our lab group was stunned by our results. The following pictures are what we saw the next day under UV lighting:

The glowing E. Coli under ultraviolet light. We have a large culture here, effectively telling us that the lab was an overall success.

A different culture with even better growth than the previous one. This culture happens to include the E. Coli that were antibiotic resistant to the Ampicillin

E. Coli alone in nutrient broth

E. Coli in nutrient broth with ampicillin. We cannot expect quality growth in this culture from bacteria without the inserted plasmid to make them resistant to ampicillin.

Data

Here is our math to determine how well the bacteria altered its genome to incorporate the new pGlo plasmid

Discussion

Our E. Coli pGLO transformation lab had the best results out of any lab we have done this school year. Plentiful colonies of E. Coli were produced on the control LB plate for both the wild-type and recombinant pGLO E. Coli. In terms of recombination efficiency, it was a complete success: we had one of the highest efficiencies of any lab group. Recombinant E. Coli flourished on the LB plate with ampicillin and fluoresced on the LB plate with ampicillin and arabinose as expected:

 The wild-type E. Coli, as expected, did not grow on any LB plate with ampicillin. Even on these empty plates, there was very little contamination. 

We contribute our success to our meticulous following of lab procedures. We used the correct amounts of materials and were not lax when it came to correct timing for the heat shock. The transfer from cold to hot to cold was immediate and precise. Our results confirmed our hypothesis perfectly: naturally, the antibiotic resistant E. Coli would grow in an ampicillin rich environment, while the wild-type would not. Overall, we are very pleased with our results.   

Conclusion

By shining ultraviolet light on the petri dish and seeing E. Coli glow in the dark, we had visual proof that the plasmid had been successfully incorporated into the bacteria cells. In addition to this, only bacteria with the inserted plasmid could grow on the ampicillin culture, which also tells us that this population of E. Coli is resistant antibiotics. This lab is designed to show how genetic engineering works and the fascinating results following the simple insertion of just one new gene to the bacteria's genome. This practice is something scientists are studying on a much larger scale with the hope that humans may find additional benefits from using restriction enzymes to alter the human genome.

References

http://www.cliffsnotes.com/sciences/biology/microbiology/microbial-genetics/the-bacterial-chromosome-and-plasmid

en.wikipedia.org/wiki/Heat_shock

http://www.jove.com/science-education/5059/bacterial-transformation-the-heat-shock-method

Restriction Mapping Lab

Purpose

     In this experiment, we separated DNA fragments by the size of their base pairs and analyzed the digested sites where restriction enzymes cut. This helped determine the number of cut sites for each restriction enzyme and their positions next to each other. We determined the total size of the DNA strands by adding up the sizes of the fragments from each digest. Since we knew that smaller DNA fragments migrate quicker than the larger ones, we were able to use the data for gene mapping.

Intro

     Molecular biology techniques often include the use of restriction enzymes to digest DNA as well as the separation of DNA fragments with the help of agarose gel electrophoresis. These techniques can be used for gene mapping and even for studying human genetic diseases. Restriction enzymes are enzymes that have the property to catalyze the cleavage of certain DNA molecules at specific base sequences. They are used for chromosomal mapping and also for gene splicing in recombinant DNA technology. Gel electrophoresis is a method used in labs to separate DNA, RNA, and proteins by their molecular size. In the process, the molecules are separated by being pressed through a gel (often agarose gel) by an electrical field. A negative charge is applied so that the molecules move towards the positive charge to be analyzed. 

Methods

The first thing that we did was use a needle point pipet in order to load the DNA into the gels, the first column contained the pMAP or lambda (“clear”) which will act as a marker within our results. Skipping the second, the third column contained PstI (“blue”), the fourth PstI/SspI (“red”), the fifth PstI/HpaI (“white”), and the sixth column with all three PstI/SspI/HpaI (“yellow”).

A picture of our group's gel; GO GO GO!

After placing the DNA into the wells (which was actually harder than it looks… at least for our lab group…), we closed the top of the electrophoresis chamber and turned on the voltage. As a result, the DNA should move from one end to the other positive side of the electrophoresis apparatus.

When we came back the next day, the gels were stained in order to make it easier to see the marks on the gel.

Graphs and Charts

Our picture of a plasmid with the restriction enzymes in their approximate location. 

Discussion

Although our results were not the best because our group had trouble placing the DNA into the gel, we were still able to draw data from the results of other lab groups. With the help of the marker lane on the far left of the gel, we were able to approximate the distance between the different restriction enzymes within a plasmid. The PstI well should only have one marker, the middle should have two and the farthest right should have three markers. By using this approximation, the distance of the strands should have been a total of 3900 and each column should have that total.  

Conclusion
The results from the gel tell us the approximate length of each band, which allowed us to visualize and the draw a full plasmid, annotated with the locations of each band along the ring-like structure. This lab is unique because we see how biology comes into play with forensic science. Comparing the band patterns of a suspect and victim creates a very powerful convition case if there is a clear match. Gels are simple to use, easy to understand and remarkably accurate, so it's no wonder that this concept has appeared on the AP Biology test for 15 consecutive years.

References
https://barnard.edu/sites/default/files/inline/restriction_enzyme_digestion_lab.pdf

http://www.nature.com/scitable/definition/gel-electrophoresis-286