Editor’s note: due to a technical difficulty, this blog is incorrectly ascribed to Tom Loughran. Its author is Paige Dausinas.
This semester our main goal was to learn about molecular biology and techniques used for experiments within molecular biology and to use different types of technology to complete them. The project we mostly learned about and what every other project branched off was from the cloning and transformation of GFP DNA also known as the fluorescent gene. GFP stands for Green Fluorescent Protein which is most commonly extracted from a jellyfish. The gene can be injected and breed into a species to project a green glow and the bacteria itself once expressed, gives off the color on simply something like a colony plate. Our objective was to successfully reproduce or clone this gene. The cloning of genes has led to the ability for insulin with people who have diabetes to have it grown based on human proteins instead of animal insulin. E. coli is the bacteria in which we clone to get this human protein for insulin that works much better than that used before. Thanks to Eli Lily and Genetech this bacteria can now be cloned and used. E. coli is used in order to clone the GFP DNA in our lab. The four main steps of cloning are PCR, ligation, transformation, and selection. I learned how to create and analyze a gel.I also read an article Francis gave to me about JAK-STAT pathway. Mainly, we did troubleshooting on the lab almost the whole semester to get it correct, and Annemarie and I learned different lab skills in doing so. First we started with PCR. PCR stands for Polymerase Chain Reaction during which hydrogen bonds are broken down and then annealing of the primers. A primer is a short single strand of DNA that binds to DNA of interest and duplicates the strand most likely. Then elongation lengthens are strands of DNA as needed.
After PCR, we take our linearized GFP and add it to a cut vector to get a GFP plasmid which has the GFP DNA in it now. For transformation, we heat shock our bacteria so it will accept the plasmid inside. Then once that is completed we begin selection. I plated our GFP and took colonies and tested to see if GFP was present. The first and probably most important technique we learned, before completing anything, was how to pipette. Learning to pipette properly is extremely important in doing each of the procedures I learned. As I started PCR, I learned how to run, create, and analyze an agarose gel containing our PCR product. The gel separates into different bands so we can see our DNA clearly and tell if the PCR was successful or not. After our first attempt we saw that we had not accurately completed PCR and we began troubleshooting. We then nano-dropped our DNA template. The nano-dropper tests for DNA present in our samples, if we see a peak at 260 then we know that we are on the right track, if not, we must start again. Any other peaks on our graph show either proteins or contamination. By looking at our results from a nano-drop after we extracted DNA and vector from a gel, the results show that our DNA is right about where we want it with a peak at 260. The nano-dropper is extremely helpful in helping discover whether your solution has DNA and if it is pure or not.
Another helpful technique we learned was how to digest our product using different enzymes. Our most successful digestion, after ran on a gel, was from November 2. We digested pGLO in restriction enzymes BamH1 and Sma1 and digested pGFP in Ecor1.
The two ladders separating the three different digests are labeled by 1Kb markers. The Ecor1 and Sma1 are both at 5Kb and BamH1 is at about 4Kb. We were looking for about 5Kb because the vector is about 4 and the GFP should be about 1Kb. And to figure out which enzymes we wanted to use we looked at a vector map or a GFP vector. It lists different restriction enzymes to use for cutting and making our GFP sequence linear.
As mentioned about we were shooting for about 5Kb of our plasmid and we figured that by looking at the vector map. Also by looking at the vector map you can see how many sites the enzyme cuts at and which ones will work best for the desired cut. Then for digestion, you have to know which buffer will work best for your enzymes and it must be very specific. Throughout the whole process of completing this lab we referred to this map many times to look at different enzymes and even to create our own primers for Hind111 Nhe1 and Sma1. The goal for primers is that you have between 40-60% C and G nucleotides. By looking at the sequence of each we discovered which would work better for our experiment. Then after successfully completing PCR we moved onto ligation.
Ligation again is the process in which the linearized GFP and cut vector come together to create GFP plasmid. We ligated our GFP back into a cut vector and ran a gel to see if it was successful. Then we learned to extract the DNA from the successful gel and nano-drop and religate it to see if it truly works. For gel extraction we used a QIA gel extraction kit. After completing this we should have both pure DNA and vector. First you have to cut the vector and DNA strip away from the gel not getting too much or too little. Then you have to add for every 1 volume of gel, 3 volumes of QG Buffer. Then next is vortexing and the whole time the solution must stay a yellowish color in order for it to work. After mixing a bit, the solution must be incubated for ten minutes at 50 degrees Celcius. After incubation, isopropanol is added and mixed. Then after the gel is completely dissolved, you must centrifuge for a minute and discard the waste 2 times adding QG Buffer each time to purify the DNA and vector. The third time, PE Buffer is added and then centrifuge again. Then to completely elute the DNA, EB Buffer is added and centrifuged. The less that is added the more concentrated the DNA should be. To make sure our extraction was correct we nano-dropped the two samples with a picture of the results above.
After ligation we did transformation. We followed a protocol for transformation of our plasmid with E. coli bacteria. The process involved 8 different steps ending with the heat shocked newly transformed bacteria shaking over night held at 37 degrees. After transformation is completed, selection and plating comes next. The plates used for this lab was an ampicillin plate. I learned to create an ampicillin plate and how to plate our bacteria. The bacteria is placed in the center of the plate and then spun and distributed equally around the plate. It is then held in incubation so that the bacteria can grow on the plate. After plating is complete and the bacteria has grown, under a UV light, the fluorescent green colonies that successfully up took our GFP DNA can be seen and observed. You can also spot the colonies that grew but did not have GFP in them. And once you have your successful colonies you can collect some of the flourescent ones and draw on a plate using a pipette tip.
This plate shows bacteria expressing six different types of flourescent proteins
Our project was finished after completing all four of these steps along with learning digestion of enzymes, creation of a gel and ampicillin plates, how to nano-drop and analyze the results from a nano-drop and gel.
Francis showed me and article about the JAK-STAT pathway which is responsible for the turning on and off of certain processes inside a cell. JAK stands for Janus Kinase and STAT is for Signal Transducer and Activator of Transcription. The article discusses how SOCS is turned on and off within the cell and how the STAT 3 dimerizes which means it combines with another STAT and can then enter the nucleus. A kinase is a phosphorate and a phosphotase de-phosphorates within the cell. I also learned that STAT 3 is a transcription factor, which means it can turn on and off multiple genes in the nucleus. If this pathway is disrupted or functions improperly, scientists have discovered it can cause cancer and immune dificency syndromes in the human body.
In learning and using MATLAB, a computer programming software, I plan to use and apply it to my biotechnology studies. By using a histogram, I could bin nucleotides and by writing a simple function predict how many strips of replicated DNA can be made in PCR based on time and make a simple graph using the data. I hope to write a program that would be useful for predicting the average outcome of bacteria on a fully grown plate and chart my data. Learning to write functions and create different types of graphs along with just learning some of the code was very useful for combining our MATLAB skills along with my biotechnology studies.
Next semester, I am hoping to help Francis and Aprell with whatever projects they plan on giving us. And learn to completely new techniques and information along with strengthening the lab skills we learned this semester considering we ended our current project at the end of semester.
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