Normally we only have 5 days to complete the GENO activities, but this time we were given 10 days. However, we only used 8 days. We were able to complete all GENO steps, including PCR, ligation, transformation, plating and painting. And, on the last day we gave a challenging quiz as usual.

We have done GENO several times in the past 2 years at Washington High School, New Prairie High School, St Joseph High School and Trinity School. But the last times in the past couple months at WHS and Trinity, I feel I really knew the lab. Not that I knew the science, but that I understood what to teach to my audience.

For example, at WHS we had freshman honor biology students, whereas at Trinity we had advanced sophomore chemistry students. And back in December, we had AP biology junior students at New Prairie. Not all of these students can understand the same level of material in just a weeks time. So, we have to pick and choose what is most important for them to know at their learning level (i.e., each group had different backgrounds in biology material).

I also learned how to make the lab flow smoothly in time management. At both Trinity and WHS we had some short days, but we still were able to get things done. And, even if we had normal class time, we still could break up the time to do things efficiently. Students did not have to get a pass to their next class because we ran over (this happened at St. Joe in our first GENO trials).

I am not perfected in my methods in any way…but I do feel more equipped to enter the GK-12 classroom. The only thing I am sad about is that I no longer will do this thru NSF or NDeRC. Wednesday was our last day. However, as I prepare for a possible teaching career next year (i.e, college level), I know that the NDeRC GENO experience has given me a big step forward.

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.

NANODROPPER: Check Concentation

The research students learned how to use the nanodropper (in the biology department) to measure DNA concentrations. This is a very specialized spectrophotometer instrument that measures the absorbance of a solution using a particular wavelength of light. The absorbance readings are translated to concentrations (i.e., nanograms/microliter). Different absorbances/wavelengths are used for different substances (e.g., DNA, RNA, protein, etc), and you only need 1-2 microliters to test a sample. For more info check out this link: nanodropper. We used an absorbance reading of 260nm for DNA.

To our surprise, there was no DNA in the solutions sent to us last month. This could of been due to various reasons, such as poor preparation or denaturation over time in storage or shipping. Nevertheless, the Grad Fellows used this as a teaching opportunity to show the high school research students how to prepare more DNA template. Note the DNA template used in PCR is the GFP plasmid.

MINI-PREPS: Make more GFP plasmid

I had some frozen bacteria in my lab that were already transformed with GFP (-80 degrees Celsius freezer). I streaked an arabinose-ampicillin agar plate with very small amount of frozen culture, and the next day had small GFP colonies on the plate. I used a sample of just one tiny colony to inoculate 5mL of culture broth. This is called a “mini-culture.” I made 4 mini-cultures total. The cultures were allowed to grow overnight at 37 degrees Celsisus, in a shaking incubator (i.e,. the tubes were vigorously shaken). This produces the growth of millions of bacterial cells, that will all have GFP DNA plasmid. Thus more bacterial growth equals more plasmid.

The next day, I centrifuged the cultures, obtaining a bacterial pellet. The research students then used a “mini-prep” kit to lyse open the bacterial cells in order to get the GFP plasmid. The plasmid was then purified, and nanodropped again.

For each mini-prep, we were able to obtain 50uL of DNA plasmid. Using the nanodropper, we found that each prep had a concentration of about 90-100 nanograms per microliter. This is 100 times better than our original tube of template! We then used these preps for our PCRs, and did obtain positive results.

I felt that most of the attendees that came to our ND exhibit were middle schoolers or younger (all the way down to 4 year olds). There were some high school and college visitors as well. It was definitely quite an experience to share the GENO project with not only the students, but with their parents and teachers. I met a couple scientists from different companies that had worked with GFP (green fluorescent protein) in the past. I even met one older scientist who use to catch Jellyfish so many years ago for the GFP protein.

I was able to share this project with others at so many levels. For example, a high school student or even a middle schooler may have understood the DNA, vector and transformations. They had some background in DNA genetics (or the expression of genetic traits). But, for the small children, we mainly talked about the comparison between GFP fluorescence and jellyfish bio-luminescence.

It was a long day (9am-5pm for me), but I am glad I was able to be a part of it. I did feel that the turnout for this free public event in Indianapolis was not as high as it could of been (being a metropolitan area). But, maybe next year, the event would definitely grow more.

So, I decided to make some cross-sections of fixed embryos/larvae, to see if the fluorescent labeling method would be better. The problem with sectioning is that the embryos/larvae are so small. You have to position them just right to get good orientation in sectioning. But, if you section enough, you will get a few really good sections. John G. did the immunolabeling of the cross-sections.

Here are images of the tyrosine-hydroxylase labeling of the dopaminergic cells in 3 day old larvae (green fluorescent color).

These are normal (non-mutant) fish. The signal is clearer, and there is minimal background. Note the labeling in the eyes and brain (area between eyes), and some cell axons. The cells bodies are bright green. Depending on the cross-section, you will get good orientation of the retina in the eye (with lens). Not every cross-section is good.
I sectined ~15-20 larvae to get these pics. (I put about 10 larvae in an ice block, and section all of them at the same time…takes me about 15 min per block).

I think it has been a good week. Today, the teachers saw their GFP paintings. Everyone’s worked except for one, which was probably due to painting with a negative colony (non-GFP).

So, all the supplies are going back to the Jordan stock room until the late summer. But, during the summer the Bio-grad fellows are going to think about how to make the GENO lab more efficient in the classroom. We got a couple new ideas, such as making pre-and post-quizzes.

This idea was suggeset by John Gensic who uses google apps often for students to take quizzes and tests on the computer. He then can see how the students answered on each question, and identify the problem areas. This week, we got a chance to witness this process when the Bioeyes teachers had to do a 10-question pre and post quiz for the modern lab. The post quiz definitely showed that the teachers now understood some of the vocabulary and techniques for the lab, and we could identify the topics that still needed attention during our discussion.

Imagine how this tool would be so useful in the high schools! Since we are only in the school for 5-7 days, we are sometimes not sure if all the students grasped the concepts. We do give them a quiz at the end (paper copy), and go over the questions. But, the computer method may be a good way to collect “data,” on what was learned…and to also have instant grading (not checking things off with a red marker during the class time).

ok…it’s time for lunch:-) i’ll be back later to blog!

The teachers got to see their transformations from yesterday. 3 groups had GFP bacteria, but there was so much growth that the whole agar plate looked green! So, I brought in one of my control plates, and had them pick a GFP colony to make the paint for their paintings. We’ll see the works of art tomorrow!!!

Also, Francis had a good idea today for the teachers to look up GFP in the news (using google search). They then had to blog about it. I’m going to try to maybe do this myself this week…and learn about some new techniques using GFP. This will also be a great exercise for the high school students too…i.e., they could do their own search or we can pick out articles for them to read. (note that these are news articles versus scientific journal publications).

The lab is also creating a red glowing substance from soybeans.  Below is an exerpt from the article that I thought was very interesting!

In addition to its work with GFP, Brighter Ideas is studying the use of soybean peroxidase (SBP) as an industrial replacement for formaldehyde. “We have developed a unique method for purifying the protein—patent pending—and we have found two other applications for SBP that are also patentable but not ready to be disclosed to the public at this point,” Ward said. He enjoys working with both GFP and SBP for aesthetic reasons over and above their scientific usefulness, explaining, “Most proteins are colorless, but GFP is brilliantly green and SBP is brilliantly red. Every day is Christmas in our lab.”

Interesting short article about what we have been creating GFP in the lab.  We have talked about vectors and I actually understand some other articles I have read.  The world of science is very interesting and we actually created GFP in the lab on the ND campus.  This has been a rare opportunity for an educator like me.

http://www.thesuitmagazine.com/technology/science/21209-brighter-ideas.html

The success so far is that we had 7 of the 8 PCRs work! We had the teachers try the traditional way of adding each reagent (Buffer, dNTPs, Taq, Primers, DNA, etc), and we also had them do a PCR with a commercial mastermix that already included most of the reagents. Both ways worked, however, the mastermix PCR produced much more PCR product. (We are considering this for future GENO weeks).

This morning, we did the ligation and transformation steps. Normally, this would be spread out to two days, but we had a “double lab” due to lack of time. Tomorrow, we’ll see if everyone’s transformations worked. One thing we did differently is that we put the transformed cells in a shaker (my lab), and shaked the cells at 200rpm for two and half hours. This speeds up the growth of the transformed cells, which normally grow slowly overnight without shaking. After the shaking, we plated the cells onto agar plates.

The community goes beyond the modern lab though. Lunch has been a great time to talk with the Bioeyes participants. I’ve learned that one teacher has a daughter that does modern lab work, and she is now asking her daughter more questions about her DNA work. I’ve learned investment strategies from another (stock market), and the workings of fifth grade education from another. And, I’ve been able to share somethings with others about graduate life and research. It’s been quite a diversity, but the connections made this week have been great.

I like the small group atmostphere, and I’m wondering how to incorporate that into future GENO works. Although it’s normally one grad fellow for 12-25 students in the classroom during the school year, we may can have team leaders for different sections of the class..and have the class learn in groups, rather than sitting with the whole class. I know that another university does this for their GK-12 project (i.e., assign team leaders), They invite the “team leaders” to the university to see the lab, prior to the classroom project. Something to think about…