Final+Project+Draft

A field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose. Biotechnology draws on the pure biological sciences (genetics, microbiology, animal cell culture, molecular biology, biochemistry, embryology, cell biology) and in many instances is also dependent on knowledge and methods from outside the sphere of biology (chemical engineering, bioprocess engineering, information technology, biorobotics). Conversely, modern biological sciences (including even concepts such as molecular ecology) are intimately entwined and dependent on the methods developed through biotechnology and what is commonly thought of as the life sciences industry. Throughout this semester, I have been introduced to the fundamental techniques needed in analyzing DNA by working with the Cloning a Fluorescent Gene (CAFG) Lab. I have gotten an in-depth look at the lab and how it works. Cloning is a process used often in biology and genetics in order to genetically engineer proteins and organisms. The following is a basic outline of CAFG: Objective: Clone a green fluorescent gene (GFP) into an E. coli bacteria that will express the gene and produce protein.
 * __(This will be transfered ‍‍‍‍‍to the Geno page ‍‍‍‍‍ ‍‍‍‍‍when complete ‍‍‍‍‍.)__**
 * Biotechnology: **
 * Cloning a Fluorescent Gene: **

1.PCR (Polymerase Chain Reaction)
 * DNA template and other reagents are combined to create millions of copies of the GFP
 * 2. Ligation
 * Insert the linear GFP into a linear vector to make a circular plasmid. ||

3. Transformation and Plate
 * Plasmid is inserted into E. coli bacteria.
 * Bacterium is plated on agar plates.


 * 4. Selection
 * Bacteria with GFP protein are selected. ||

**__My Experience with Ligation:__** The plans for next semester are undecided yet, but we currently talking about maybe jumping into one of Aprell's labs that she is doing. She is using a protein found in rats and using a process in which she knocks out the protein or causes the gene it comes from to no longer be expressed. In doing so we are looking into what could affect cell growth. I am interested in it because it uses the same techniques that we learned this past semester, but for different applications. For instance, she starts with a protein and then works backwards to get a gene in order to try stop the gene from producing a protein. I am excited to learn what else I can do with the skills I have acquired over the past semester.
 * Why do each step? **
 * __PCR __**
 * The goal in this lab to clone a protein. The first step in doing this, the step is already done for us before starting the lab kit, is to isolate the gene that will cause the expression wanted in the protein. When that gene is found, we must extract it and make many copies of it so that we will later have enough DNA to grow colonies.
 * PCR is the process used to make all of those copies.
 * __Ligation __**
 * In order to make protein, we first need a full plasmid to work with; a single short sequence of DNA like our GFP will not be able to because we need a circular strand that allows continuous processes.
 * Using plasmid guarantees that only the trait we desire is expressed in our protein that we are cloning.
 * __Transformation __**
 * After we have replicated the gene and then inserted that gene into a plasmid, we need the DNA sequence to cloned and made into a protein using mRNA. Usually, the protein is made in its environment, in this case the jellyfish that the fluorescent green protein comes from, because the living environment is conductive of the process. After PCR and ligation, we only have plasmid outside of a living organism. So now we heat shock the E. coli to insert the plasmid in the bacteria.
 * Now the bacteria are left to do the rest of the work creating a protein to express the gene we want.
 * Then, we plate the bacteria to grow colonies of protein that contains the GFP gene.
 * __Selection __**
 * Only protein without contamination and containing the GFP will survive the ampicillin and arabinose covered agar plate. Our GFP is ampicillin resistant, whereas other bacteria are not. And our plasmid needs the presence of operon AraC to grow colonies.
 * Explanations of Steps: **
 * __PCR__**
 * Reagents Needed: Template DNA, Forward Primer, Reverse Primer, dNTPs, PCR Buffer, Enzyme, Water Taq Polymerase
 * The reagents are added to a PCR tube with a pipette. Then, the tubes are then placed into the PCR machine to go through cycles of amplification that allow for millions of copies of the template DNA to be created.
 * After the PCR is done, ‍‍we use an Agarose Gel Electrophoresis to verify if the results are sound‍‍.
 * A gel electrophoresis, or simply running a gel, is simply a way to measure products according to the size of the fragments. The PCR products are inserted into grooves at one end of a square block of gel about a centimeter thick. The gel is then placed in a machine in which the it is submersed in buffer that conducts electricity. Charges are attatched tp either end of the machine. DNA is negatively charged so when the positive charge is attatched to the side of the machine closest to the grooves containing the PCR products, the products are pushed toward the other side causing them to travel through the gel. Larger fragments experience more resistance than smaller fragments. We are able to tell the exact size of the fragments because we also load a ladder into one of the grooves in the gel. The ladder works as a kind of ruler to measure the size of each fragment band that shows up in the UV photo of the gel.
 * Good PCR results for PCR show one line at about the 1 kb mark.
 * https://sjhsrc.wikispaces.com/PCR
 * **__My PCR Experience:__**
 * This semester was a lot of troubleshooting in the lab, starting with PCR. This was a set back sometimes, but it allowed for me to get a better understanding of each step in the CAFG lab. There were many times that we were dealing with old or poor quality DNA. In these cases we had to use DNA from previous labs that was frozen and kept properly, or as we finally did, we ordered plasmid DNA from a well-established company.
 * __Ligation__**
 * DNA ligase is used to join together a linear vector and the linear GFP. This forms a DNA plasmid which is circular. The plasmid is different from normal bacterial chromosomes because it only contains the specific gene that the researcher wants.
 * The way this happens is the double strand GFP and vector are denatured, pulled apart by heat, then using the ligase that attaches onto sites where vector and GFP will join, with the primers the DNA is annealled, or glued back together into a plasmid.
 * https://sjhsrc.wikispaces.com/Ligation
 * https://sjhsrc.wikispaces.com/Ligation
 * Ligation was not a step that usually gave us trouble. Once we got past our DNA and PCR problems, we usually were okay. However, we did have trouble one time where our control Ligation produced plasmid DNA but our experimental reaction did not. However, in this run that we did, the ligase was our variable, because we were testing the new ligase we had been sent. Therefore, we were able to know that the ligase was faulty because it would not anneal the plasmid.
 * __Transformation and Plating__**
 * <range type="comment" id="587170">‍‍‍‍‍Cells do not normally allow DNA through their membrane, as a protective mechanism. To trick the E. coli bacteria cells to take in the GFP, we have to shock the cells into panic so that they become chemically competent, meaning that heat was the cause of the bacterium taking in the DNA around it to try to survive, including the GFP plasmid. ‍‍‍‍‍
 * After this process is over, we have bacteria containing the GFP that will grow colonies of our GFP protein.
 * The bacterium is then plated on ampicillin and arabinose covered plates. This allows us to know that the only bacteria on the plate is that with the GFP in it, as the GFP is ampicillin resistant and requires arabinose to produce the desired protein.
 * https://sjhsrc.wikispaces.com/Transformation
 * __Selection__**
 * The DNA in the bacteria has been transcribed and then translated in the protein we need, which is then selected and activated by chemicals on the plate to grow colonies. First, the GFP is ampicillin resistant because of the Ampr gene, and the plate is coved with ampicillin. This way, almost all of the other DNA or bacteria on the plate will be killed, so that it does not grow in colonies on the plate. Second, the protein that we need requires the inducer arabinose to trigger the AraC operon that regulates the protein polymerase.
 * When the colonies have grown, all of them should be fluorescent because we have successfully cloned the GFP gene into a protein.
 * __My Experience with Selection:__**
 * Selection is part of the cloning process that you would completely miss if you didn't know that something else was happening. The only way to know it took place is to run a black light over the plate and see the glow-in-the-dark green.
 * After all the science is over, you can select a single colony, which is the size of the tip of your pencil, pick the colony up with a pipette tip, put it in buffer and use it as paint to draw on plates!
 * Challenges Faced and Troubleshooting: **
 * __PCR__**
 * What do you do when the PCR does not produce results?
 * The gene to be replicated may not be good. In this case, we may simply have to use different DNA for our PCR. DNA, if not stored properly or if it is too old, may not react properly in PCR.
 * Taq polymerase is the catalyst in the PCR reaction. If the PCR does not produce any results, it could be an effect of faulty or too low of a concentration of taq. To determine this, do more PCR reactions using different concentrations, and also using different taqs. When this happened in our lab, we used the old Geno taq that was left, the new taq that had been sent, and Aprell’s taq.
 * What if you run out of template DNA?
 * When cloning the GFP gene, especially if there are many complications, it is easy to run out of DNA. When you start to come to the end of your supply, this is a sign that you need to make more. First, mini-prep the old DNA; this copies the entire DNA sequence. Finally, do a do a digest; this cuts the specific gene to be cloned, the GFP, out of the DNA sequence. When the digest is done, you will be left with a new supply of GFP template DNA for PCR.
 * https://sjhsrc.wikispaces.com/Mini-Prep
 * https://sjhsrc.wikispaces.com/Digest
 * __Ligation__**
 * How do I know the ligation worked?
 * If the ligation worked properly, you no longer have a vector and a GFP, you now have a plasmid. In order to verify this, run an Agarose Gel Electrophoresis. The result you are hoping for when you photograph the gel is three bands close together toward the top of the gel. However, if the ligation did not work, you will find two bands in the photograph, one at the 4kb mark and one at the 1kb mark.
 * What if the ligation did not produce the proper results?
 * This could be a result of faulty ligase. When this happens, the only thing to do is find another ligase or order new.
 * You could need a higher concentration of DNA or vector. To determine the concentration of each of these, use a nanodrop machine and software.
 * __Transformation__**
 * What if the transformation and plating does not produce and colonies?
 * If you have gotten to this point without problems, there is nothing wrong with any of the DNA or the ligation process. The only thing to do is redo the heat shock process to guarantee that nothing had gone wrong in the first trial.
 * __Other__**
 * How do I verify that the plasmid DNA is what I think it is?
 * When ordering DNA, you are usually sent a vector map and sequence. Using the vector map and online data bases such as [] to find the codes for restriction enzymes. Using both of these you can find the best restriction enzymes that should cut out the GFP. If you run a digest and photograph the gel, you should find the two bands: one at 4kb and one at 1kb.
 * If that is not what you find, the DNA is not correct for this experiment.
 * *(using the DNA sequence, you can also design your own primers from the restriction sites)
 * Plans for Proceeding Next Semester: **