NDeRC Genetics collaboration blog

During the past 2 weeks, Francis Raycroft and I did the last GENO week at Trinity School at Greenlawn. We had a total of 26 students (17 girls and 9 boys). Their teachers were John Lee and Erica Price.

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.

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By the efforts of the St. Joe research students, we were able to start trouble-shooting the CAFG lab. A couple weeks ago, we found that none of our PCRs worked. Our first approach was to check the concentration of the DNA template used in the PCR.

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. (more…)

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This past weekend, I attended the Celebrate Science Indiana event at the state fair grounds in Indianapolis. The event included several science presenters (i.e., universities, agencies, etc) that each had a booth for their exhibit. NDeRC had a booth, along with the ND chemistry/physics department. We brought Bioeyes, NANO, and GENO. I ran the GENO exhibit, displaying several petri dishes of fluorescent bacteria (in protective glass of course). The students could shine the black lights across the petri dishes to view the fluorescence. I also had a poster that explained the gene cloning techinique.

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.

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This week, Francis Raycroft and I met with the St. Joe High School research students to discuss this month’s project. The two students (AnnMarie and Paige) will be going through the GENO lab and learning basic molecular biology techniques. On this past Wednesday, we did an overview of the entire lab (PCR, ligation, transformation and selection). On Monday, we will begin with the PCR lab. What makes this experience different from previous GENO labs is that we will be able to do extra molecular techniques that we cannot do in the high school (i.e., because of time)…for example, we may do some gel electrophoresis. And, if certain steps do not work in the lab, we will be able to go back through them and optimize the protocol several times. The two goals from this month is to teach the students molecular biology, and to really optimize the GENO lab methods. And of course, this may also be part of October’s goals.

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John G. joined the lab a few weeks ago to work out the whole-mount immunolabeling of our wild-type and mutant fish. Thus far, we have had problems with too much fluorescent background, mixed with our specific signals. We see the dopaminergic cells, but we also see everything else in red color (although dimmer than the color in the dopaminergic cells).

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).

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….so it’s Day 5 of Bioeyes (the last day), and the teachers are now filling out the surveys. I don’t have a survey, so I am secretly blogging at one of the computers….

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!

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It was Day 4 of the modern lab for the bioeyes institute.

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).

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Today is the third day of the Bioeyes Institute 2011. It’s a small group this year (~7 teachers), but it’s been a good week. I have been working with the modern lab, cloning a fluorescent gene (the GENO project). It’s been kind of beneficial to have a small group…the discussions are good, the lab goes smoothly. All this could be due to the fact that we (the grad fellows) have one year experience with GENO. However, the small group atmosphere has essentially allowed for “one-on-one” assistance during the lab, and everyone is somewhat “connected,” like a small community of modern scientists.

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…

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I’ve realized that it’s been way too long since my last post…probably because I had a committee meeting last month that kept me busy in getting a presentation together.

But, I’m back!!!

And the summer is starting out good. John Gensic has joined our lab for the summer, and is starting out on a project using zebrafish embryos. He is doing whole-mount labeling of the zebrafish embryo. This means that he will not have to make cross-sections of the embryo, but will be able to see fluorescent labeling by looking at the “whole” embryo. This requires bleaching the embryo (to remove the pigment so we can see through tissues), and permeabilizing it to make sure the labeling can get “inside” the body. He will be doing fluorescent immuno-labeling of dopaminergic neurons using an dopaminergic-specific antibody for tyrosine-hyrdroxylase (TH) and a fluorescent secondary antibody.

The specimen of interest: night blindess b (nbb) mutant zebrafish embryos. These embryos have a mutation in the STIL gene. This gene is also present in mammals and humans, and is known to be involved in cell growth. We want to understand it’s role in neural degeneration.

The question: Do the nbb mutant embryos show a loss of dopaminergic cells during development? And is there a difference in this loss between heterozyous and homozygous mutants? (i.e., Does a mutation in the STIL gene affect dopaminergic cell development?)

The hypothesis: Because heterozygous adult mutants show some loss of TH cells in the retina, there may be a problem with dopaminergic cell development in the heterozgyous embryos (which can grow to adult), and an even more severe loss of dopaminergic cells in the homozygous mutants (which only live 7 days).

The method: Fluorescent whole-mount immuno-labeling. Collect mutant zebrafish embryos (2-3 days old), remove pigment, and permeabilize them. Then, label dopaminergic cells in the retina and brain using the tyrosine hydroxylase (TH) antibody. (Dopaminergic neurons release the neurotransmitter dopamine that can affect neural processing such as vision. TH is an enzyme involved in the synthesis of dopamine. Thus, dopaminergic cells contain TH, and can be labeled by a TH antibody.) This TH antibody will bind to the enzyme in dopaminergic cells, and a fluorescent secondary antibody can be used to bind to the TH antibody. Thus, we will be able to see fluorescent dopaminergic cells in the embryo.

Results….well, here’s one photo of a normal fish with TH labeling in the eye (you can see a “ring” of cells” around lens of eye)…still working out the microscopy photo conditions to make the fluorescent signal brighter….(you’ll have to click on photo to see up close).

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So, it came to my attention earlier this month that the material for GENO week is sometimes too advanced to cram everything in 4-5 days. After attending the GK-12 conference, I learned that some projects are in the classrooms for weeks at a time (not days). Our project is accelerated in the sense that students have only a week to grasp these modern biology topics.

Although we have powerpoint…and molecular biology supplies…and lecture notes…and fluorescent bacteria….I still felt we needed something more for the students to understand what they are doing. So, I made some preliminary worksheets for students to fill out as they do the lab manual reading.

The idea was that they would work on these short-answer questions the night before when they would actually read the lab chapter. We have worksheets for chapters 1-3 (Pipetting, PCR and Ligation).

The first school to try this was Washington this week, a freshman honors biology class. They got chapters 1-2 to work on over the weekend before lab monday, and chapters 3 homework for tuesday.

After “grading” the first 2 sets (pipetting and PCR), I was amazed that the students for the most part did answer the questions. Most were correct, while some were left blank or had wrong answers. But, at least they tried, and at least we know the problem areas. We went over some of the questions in the beginning of class.

No handout was given for Wednesday because we felt the “paper work” may be a bit much (they have class test on Friday). And, guess what? The students did not read the lab manual last night. Thus, the worksheets do help to push them to read.

Today (Wednesday), I made a short fill-in-the-blank handout for the transformation lab. Basically, they had to read through the glossary in the back of the manual to match the selected terms with the definition. I let them silently work on this for sometime in class (7-10 min; during our 20-30 min wait time for the lab). And, they worked hard to fill out the worksheet. We went over the answers.

So my conclusions are the following:

1.) More students (not all) read the lab manual when they have a worksheet
2.) Worksheets can help target problem areas in learning (because most of the time students don’t say they have questions.)
3.) Worksheets can provide a different activity other than lecture during lab wait times.

But, after grading, I did see the need to change the wording on some questions…and thus the handouts are still a work in progress. And maybe later we can come up with some games or other activities to make the learning process more enjoyable.

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