Bio@Tech

Here are the group photos for the July 2011 session of Bio@Tech. Thanks to Mr. Li, Irwin’s father. And thanks to all for a great 3 weeks!

The rambunctious crew

The crew with Virginia, Dr. Green and Dr. Choi

Click on the above photos to see them full-size.

GMO Test Gels A (left): Strawberry; B (right): Pluot

The photos of the gels from the GMO testing are attached.

Gel A: Strawberry

Gel B: Pluot

Gel C: Broccoli

Gel D: Zucchini

Recall that the lanes in each gel are:

1. Markers

GMO Test Gels C (left): Broccoli; D (right): Zucchini

2. non-GMO w/ plant primers

3. non-GMO w/ GM primers

4. test food w/ plant primers

5. test food w/ GM primers

6. positive control GM DNA w/ plant primers

7. positive control GM DNA w/ GM primers

Your mitochondrial DNA sequencing results have been received from Genewiz, the DNA sequencing company, and attached to this post as zip files. For some reason, the sequencing did not work well; all the sequence trace files show a high background and non-specific priming. I suspect that the sequencing primer may have been degraded.

The mitochondrial DNA sequence text files are in the _seq zip file, and the trace files are in the _ab1 zip file.

After you download and save these files to your computer, you should unzip the files. You can open the sequence files using notepad or any other simple text editor. To open the .ab1 files, you have to use a special viewer like ChromasLite, which is freely available (Windows only) here:

http://www.technelysium.com.au/chromas_lite.html

Alternatively, you can load and view the trace files in MEGA5. After you start up MEGA5, open the Align menu and click on “Edit/view sequencer files (trace)” – that will open a file search box where you can specify the sequence you want to load and view. That will open a sequence viewer that looks and works like Chromas Lite.

Given the lower quality of the sequences this time around, we cannot really do any further analysis. I will request that they re-sequence the samples using a different primer, and will post the results here next week.

The story of the chikungunya virus illustrates not only a newly emerging viral disease, but how evolutionary theory and DNA sequencing can be applied to understand the origin of these outbreaks and help to predict and possibly contain or prevent future outbreaks. This will also show what public informatics resources are available on the internet, and how to access them.

Read: http://www.smithsonianmag.com/science-nature/The-Next-West-Nile-Virus.html

What technical terms or parts of the story would you like further clarification or explanation?

What is an alphavirus?

What is a togavirus?

What is the genome of the chikungunya virus – DNA or RNA, double-stranded or single-stranded?

For single-stranded RNA viruses, what is the difference between the (+) strand and the (-) strand? What difference does it make in terms of viral replication?

The story mentions studies by scientists about mutations in the virus. How can you find the actual papers or citations for these studies? List the citations to these papers, if you can find them.

What more do you want to know about this virus? List your questions.

Where can you search for information to answer your questions about this virus and the disease it causes?

Where have outbreaks of this virus occurred, and when? Map the outbreaks on a map of the globe.

Is this virus continuing to evolve? What strains have been responsible for recent outbreaks?

A comparison of viral genome sequences, from the original, endogenous strains in Africa versus the strains causing more recent outbreaks outside Africa, can reveal how the virus is evolving, and what mutations are prevalent in the more recent outbreaks.

Search NCBI’s Genbank repository for Chikungunya genome sequences. Retrieve sequences for the virus isolated at different locations and different times, in FASTA format, and save them as plain text files using Windows Notepad or other plain text editor.

Start up MEGA 5, and open “Edit/Build Aligment” and select “create a new alignment”. The program then asks you whether you wish to create a DNA or protein sequence alignment. After you choose “DNA”, then a blank “Alignment Explorer” window is created. Import the sequences you want to compare from the “Edit” menu, “insert sequence from file”. You can browse over to where you have the sequence text files and import them.

After all the sequences you want to compare have been imported, and are visible in the Alignment Explorer window, open the “Alignment” menu, and select either “align by Muscle” or “align by ClustalW” – either should work just fine. In a few seconds, you’ll see all your sequences aligned, so that identical nucleotides appear in columns. This alignment allows you to see where the differences are, because the 4 bases are color-coded.

Do you see a mutation that is shared among all the more recent, non-African strains, and absent in the original African strains?

Next, you can use the multiple-sequence alignment to build a phylogenetic tree of the aligned sequences. A phylogenetic tree illustrates the inferred evolutionary relationships among the sequences. Go to the Phylogeny menu, and you see options for Construct/Test various types of trees. Explaining the various options is beyond high-school or college freshman level biology, and would take too long, so I’ll advise you to choose the Neighbor-Joining Tree. Use the currently active data (data that’s already loaded into the program). Accept all the defaults for now in the Preferences box, and click the “Compute” button. You should now see a tree. This Tree Explorer window then gives you various options to format the tree. Since we do not know at the outset which of these sequences, if any, is ancestral to the others, the best format is a radial tree.

What does you tree suggest about the evolutionary relationships among the viral strains in the different geographical locations? Did these strains all come from Africa, or from other locations?

Fore more information about Chikungunya virus, visit the CDC’s web page on this virus:

http://www.cdc.gov/ncidod/dvbid/chikungunya/ and download their Guidelines for Preparedness and Response for Chikungunya Virus Introduction in the Americas (pdf).

In the first session this summer, students set up Mudwatts microbial fuel cells with soil from my yard, and each group had a choice of amendments to the soil. A Mudwatt with no additives served as the control. The available choices for additions to the soil were:

• sucrose
• magnesium sulfate (not done)
• magnesium acetate
• magnesium chloride
• magnesium nitrate

Each group added 5 grams of one of these additives to the soil and water to make mud, and then set up their Mudwatts fuel cells with the hacker board connected according to instructions. Because of the short time available, we incubated them at 37 degrees for 75 hours. Only the Mudwatt with sucrose had a flashing LED. Nevertheless, the students performed a potentiometry sweep to determine the maximum power output from each Mudwatt. Only the Mudwatt supplemented with sucrose gave a higher power output than the control – all the others had significantly lower power outputs.

In the second session, we will do another measurement of power output from the Mudwatts set up by the first session students, and then take them down to set up new microbial fuel cells. This time we will use sediment from a creek (North fork of Peachtree Creek) and test different additives. The additives I have in mind are:

• sucrose (again)
• vegetable oil
• glycine
• beaten egg

If there are other substances/foods you would like to try, you are welcome to bring them, along with an explanation of why you think they might help.

I’ve attached the instructions and information about Mudwatts and microbial fuel cells and a sheet of questions as a weekend homework assignment (click attachments below this post). You should be able to download them as pdf documents. I’ve also attached this week’s schedule.

See you Monday!

I hope everyone had a wonderful and enjoyable learning experience this month. Here are the group photos. You can click on it to view/download a larger version.

The crew with Virginia Chu and Jung Choi

Another photo of the crew, about to enjoy the rest of their summer.

I’ve been playing with the mitochondrial DNA sequences using the software package called MEGA 5. MEGA stands for Molecular Evolutionary Genetic Analysis, runs on Windows, Mac and Linux, and is available free at http://megasoftware.net/.

With MEGA, you can view ABI sequencer trace files (filenames that end with a .ab1 extension). This viewer is located, somewhat unintuitively, under the Align menu as “Edit/View sequencer files (trace)”.You can view your trace file, and also reverse complement the sequence. That is, get the sequence of the complementary strand.

If you want to compare multiple sequences, such as all your friends’ DNA sequences with your own, and maybe throw in a Neanderthal or two and maybe chimpanzee or gorilla, you open “Edit/Build Aligment” and select “create a new alignment”. The program then asks you whether you wish to create a DNA or protein sequence alignment. After you choose “DNA”, then an “Alignment Explorer” window is created. basically a blank window. You can then import the sequences you want to compare from the “Edit” menu, “insert sequence from file”. You can browse over to where you have the sequence files, in either text form or as .ab1 trace files, and import them one by one, or select multiple files and import all the selected files at once.

After all the sequences you want to compare have been imported, and are visible in the Alignment Explorer window, you open the “Alignment” menu, and select either “align by Muscle” or “align by ClustalW” – either should work just fine. In a few seconds, you’ll see all your sequences aligned, so that identical nucleotides appear in columns. This alignment allows you to see where the differences are, because the 4 bases are color-coded.

What I found really cool about this is that the Alignment Explorer allows you to edit the sequences. Seeing the sequences aligned, I could easily select the around 40 columns at the start of the aligned sequences that were all noise and junk, and delete them. Likewise, I was able to delete all the phantom sequences at the other end. I saved the resulting alignment file and attached it to this post as a zip file (for some reason security would not allow attaching the file in its native form).

A phylogenetic analysis with this trimmed alignment still gives poor results, because 4 of the sequences are problematic after their homopolymeric G tract. A further trimmed alignment with only the columns up to and including the G tract will give much better phylogenetic trees.

Making a phylogenetic tree is deceptively easy. Go to the Phylogeny mmenu, and you see options for Construct/Test various types of trees. Explaining the various options is beyond high-school or college freshman level biology, and would take too long, so I’ll advise you to choose the Neighbor-Joining Tree. Use the currently active data (data that’s already loaded into the program). Accept all the defaults for now in the Preferences box, and click the “Compute” button. You should now see a tree. This Tree Explorer window then gives you various options to format the tree. One option you should definitely explore is in the Subtree menu: “Root”. With this you can indicate which sequence on your tree is an “outgroup” – a sequence that is definitely more distant, by other criteria, from all the other sequences in your tree. An example of an outgroup would be a chimpanzee sequence where all the other sequences are human. “Rooting” your tree will then place all your sequences in a phylogenetic tree diverging from the last common ancestor of humans and chimpanzees.

The gel photos from the GMO testing we performed today have been posted, and are also attached here. The photos are labeled gmoAB, for gels of groups A and B, and gmoCD for gels of groups C and D. On each gel, the lanes are, from left to right: marker, samples #1-6.

The samples are:

1 Non-GMO control food, Plant primers

2 Non-GMO control food, GMO primers

3 Test food, Plant primers

4 Test food, GMO primers

5 GMO positive control, Plant primers

6 GMO positive control, GMO primers

Gel A: apple

Gel B: papaya

Gel C: corn

Gel D: banana

We received the mitochondrial DNA sequences from Genewiz by email. I’ve downloaded the sequence files and trace files as zip files and attached them here.

After you download and save these files to your computer, you should unzip the files. You can open the sequence files using notepad or any other simple text editor. To open the .ab1 files, you have to use a special viewer like ChromasLite, which is freely available (Windows only) here:

http://www.technelysium.com.au/chromas_lite.html

You should use the trace file to inspect the sequence and make any corrections in the sequence text file. Everyone will have to trim off the first 35 nucleotides or so, and probably anything after nucleotide 410.

If your sequence has a polyG stretch in the middle, you will probably also have to discard any sequence that follows the string of 8 or 9 Gs.

I’ll show you how to download comparable mitochondrial DNA sequences from other people, ancient DNA specimens (including Neanderthals), and chimpanzee, and how to construct a phylogenetic tree that compares the sequences and infers an evolutionary tree.

That is the question asked in this fast-paced video:

http://www.youtube.com/watch?v=F1IWkbU0SG4

Some of the information flashes by in this video so quickly you don’t have time to reflect on them. Here is a great article in the NY Times on the looming agricultural crisis and the need for more urgency in research:

http://www.nytimes.com/2011/06/05/science/earth/05harvest.html

I don’t think we can feed the growing population with worsening climate conditions and shrinking arable land, without the toolkit of genetic engineering.