Team:Elan Vital South Korea/p process



To investigate the multidrug resistance of MRSA, we first got some frozen samples of MRSA from a local hospital. Then we raised the MRSA in liquid LB broth. Then we placed the MRSA on a solid LB plate with antibiotics, and observed their growth. Then we extracted the DNA from the MRSA via miniprep, and mixed it with E. coli, hopefully causing transformation. Now, the E. coli should have the multidrug resistance that the MRSA DNA codes for. So, we can investigate the resistance of the DNA by placing the transformed E. coli in a culture of drugs, and observing its growth. We tested the resistance of the transformed E. coli in ampicillin, gentamycin, kanamycin, and tetracycline. But before we transformed the E. coli, we placed the E. coli in solid LB plate with antibiotics and tested it for drug resistance. Without testing E. coli for drug resistance, we would not know if the drug resistance of the transformed E. coli is from the DNA of the MRSA, or from the E. coli. Now, we want to investigate the genes that were involved in the multidrug resistance. To do that, we first prepared the transformed E. coli via mini prep, and then ran the E. coli through PCR. This should multiply the number DNA in the E. coli, allowing investigation of the genes involved. Then we ran gel electrophoresis to investigate the genes involved.

We used three well-known techniques for analyzing DNA in this investigation: transformation, PCR, and gel electrophoresis. Transformation happens when a cell’s genetic material is altered through uptake of foreign DNA. Transformation happens naturally in some cases. Sometimes, bacteria are transformed without much outer interference, and viruses ‘reproduce’ by transformation, so viral infections are always accompanied by transformation.

How transformation works

Transformation was first found by a British scientist named Frederick Griffith in his experiment with Streptococcus pneumonia. In the experiment, he used 2 strains of S. pneumonia: the type III-S strain (smooth strain) which were fatal, and the type II-R strain (rough strain) which were harmless. When mice were injected with the rough strain, the mice lived, and when the mice were injected with smooth strain, the mice died. Griffith then killed a smooth strain bacteria sample with heat. When he injected the mice with the heat killed smooth strain, the mice lived. Now, he mixed the rough strain with the heat killed smooth strain, and injected the mice with the mixture. Since both the rough strain and the heat killed smooth strain were harmless, it was expected that the mixture would not be fatal, but surprisingly, the mice died. The reason for the fatalities was transformation. Heating the smooth strain caused the genetic material to fall out of the bacteria. When that was mixed with the live rough strain, the rough strain took up the genetic material and underwent transformation. Once the DNA was inside the rough strain, the DNA used the bacteria’s cell mechanism to make the toxic proteins of the smooth strain, causing the mice to die. Although Griffith did not know why the mice died, his experiment was nevertheless crucial in the investigation of the genetic material.

Griffith’s experiment

We used transformation to investigate the multidrug resistance of MRSA. We tried to transform E. coli with no drug resistance with the DNA of the MRSA extracted by miniprep. If the DNA extracted coded for the multidrug resistance of MRSA, the transformed E. coli would also exhibit multidrug resistance.

PCR (Polymerase Chain Reaction) is a technique used to amplify a certain section of DNA. PCR requires a section of DNA, some primers, and the nucleotide bases used to replicate DNA. In PCR, the whole mixture goes though many heat cycles of heating and cooling. When heated, the original strand of DNA is split in two, and each strand can be replicated separately when cooled. The primers start the process of DNA replication, so the depending on the type of primer, the section of DNA replicated can vary. That’s how we can vary the section of DNA replicated. Once the section of DNA is replicated, the mixture can be heated again so that now, the two pairs of DNA (instead of just one pair) can each be separated into two separate strands each, and the total of 4 strands can replicated independently. This process can be repeated many times so that one section of DNA multiplies into hundreds of thousands of DNA.

How PCR works

We used PCR to amplify the DNA we got from the transformed E. coli in order to analyze the DNA. It is much easier to analyze DNA in large quantities, so PCR is frequently used before DNA analysis.

Gel electrophoresis is a technique frequently used for analyzing DNA. We used gel electrophoresis to analyze the DNA we extracted from the transformed E. coli, and then amplified through PCR. Gel electrophoresis requires DNA, restriction enzymes, and stains. Restriction enzymes cut DNA at specific sites. For example, a restriction enzyme could cut DNA at ATTA. That means that whenever the restriction enzyme sees a strand of DNA going ATTA, it will cut the DNA there. That means that when DNA is mixed with a restriction enzyme, the DNA will be cut into many strands with different lengths. Gel electrophoresis relies on the DNA strands having different lengths. When we mix the DNA strand with the restriction enzyme and the stain, the DNA is cut into many DNA strands with different lengths, and each of the cut DNA strands are stained so that they are visible. In gel electrophoresis, a rectangular gel with wells on one side is placed in salt water that can convey current. Then the DNA-Restriction Enzyme-stain mixture is placed in the wells. When the water is charged, the DNA strands move across the gel because DNA is slightly negatively charged. But because the DNA has to move through gel, its progress is impaired. Remember that the DNA strands have different lengths? Larger strands of DNA have a harder time going through the pores in the gel, while smaller strands can travel more easily. Gel electrophoresis will result in different bands of DNA in the gel. The bands further along the gel are caused by the smaller strands, and the bands closer to the initial wells are caused by larger strands. Using gel electrophoresis, we can compare different DNA strands. If two similar DNA strands undergo gel electrophoresis using the same restriction enzyme and the same stain, the resulting bands will be similar (most likely the same). Even if there is a slight difference between the DNA stands, the bands resulting from gel electrophoresis will rarely be changed since only a difference in the restriction site of the enzyme could significantly change the length of the split DNA strands.

How gel electrophoresis works

We analyzed several DNA strands that we got from the cells that showed multidrug resistance. If the result of gel electrophoresis is similar, that means that the section DNA is shared in the different DNA strands of the cells. That means that that section could code for the multidrug resistance. The next natural step after this would be to extract the section of DNA and find its DNA sequence, but we did not go on after gel electrophoresis because we did not have the time.