Carl+J.+Reid

=Site-Directed Mutagenesis using GFP (Green Florescent Protein), BFP (Blue Florescent Protein), and //E.coli.//=

**__Goals:__**
toc The goal of this project was simply to determine if to determine if it is possible to change the DNA sequence and therefore phenotype of a colony of bacteria. In addition it was to learn and experience working and performing an experiment in a research lab. During my process to discover if the hypothesis was true many techniques including Site-Directed Mutagenesis were used and learned. For the project it was expected to discover that the bacteria DNA would in fact change and as a result the bacteria would glow as a result. The main purpose was to make the bacteria on the two plates both glow which would indicate a change in the DNA sequence. To reiterate the student predicted that the bacteria would in fact undergo a change in DNA sequence and thus, the bacteria would glow.

**__Discoveries:__**
In the project much was learned. First, it was learned that one can change the DNA sequence of an organism by performing simple mechanisms. This was proved by using bacteria. Second, the process of Site-Directed Mutagenesis was performed and learned. The primary purpose of mutagenesis is to create a point mutation within the sequence of the organisms. These mutations vary from deletions to insertions of single or multiple amino acids. The important concept of “Site-Directed Mutagenesis” is the fact that the mutation is created at a specific location on the DNA. How can this happen? To start, one has their DNA vector with specific inserts that the scientist desires. As well as two oligonucleotide primers, which contain the mutation of desire. DNA polymerase is added and the three of these are extended when using a thermal temperature cycler. After the process is completed one has DNA with a desired mutation. It is by the same process that many scientists insert GFP into the DNA sequence of bacteria, which make them glow. In addition it was also learned that //E. coli// are grow at very fast rates and in exponential amounts. This proved useful because the results were produced quickly. What was surprising was the fact that in the usage of BFP (Blue Florescent Protein) the bacteria were not blue but more of a white color. This was not a mutation or a problem, but this is simply the color that BFP produces. However this made me question why the protein is known as Blue Florescent Protein. This experience was beneficial and a number of things can be taken away from it. Most importantly, was the discovery of how easily one can insert a gene into DNA and change the phenotype. Also, how GFP can be used as a genetic marker and how many processes within the body of the organisms containing the GFP can be seen. This proves useful when trying to study the development of illnesses such as cancer, diabetes, etc. In the end, the hypothesis was proven because the bacteria did glow which proved that the DNA sequence had changed and the insertion of GFP and BFP was successful.

**__Challenges:__**
One challenge that was faced during the process of the project was the quality of samples used. In one case, the DNA sample used was actually mutated or deficient. The result was after preparing them and placing them into the incubator, upon taking the Petri Dish out there were no bacteria growing on the plate. This was before the insertion of GFP or BFP. This was really the only challenge that occurred.

**__Solutions:__**
The solution to this problem was the use of a Biophotometer. What this device does is it checks the concentration of the DNA by sending a ray of light through a sample which. rests in a cubette. If there is a normal amount of DNA in the sample the light amount of light shining through will not be as high. However, if there is not a lot there wlll be a lot of light that is let through. This solved the problem because it ensured that the sample used actually had DNA inside to change the bacteria.

**__Looking Back:__**
If there was more time I would have considered performing this process on bigger organisms such as Mice. It would be an amzing experience to insert GFP into a mouse because these are small steps closer to humans. In addition, mice are noticeably bigger than bacteria yet they are not massive to the point where it would take years to produce results. If I were to perform that experiment then the results would be even more useful to the scientific community.

5 Things you wish you knew before you started the project

 * 1) Try to finish everything at least a week before the project is due. This way there will not be any late nights cramming in everything and by doing this you will not be producing your best work.
 * 2) If you do the experiment make sure to do the research at the same time. Yes it may be a lot of work but this way the results are fresh on your mind.
 * 3) Try and submit a draft before the due date. The teacher will give you helpful feedback and catch any potentially major mistakes.
 * 4) Try to apply a little bit of time to thinking about what you plan on doing for your project before it has been assigned. This way, you will not stress over what to do.
 * 5) Finally, make sure you enjoy the project. The biology project is generally turned into this dreadful assignment because of the last minute cramming but make it enjoyable by choosing a topic that interests you and immerse yourself in the research and material.