Note the different echolocation frequencies! Different species of bat!
A late blog as we were out on a bat walk last night in Clonskeagh (Dublin Suburbs). Prior to the walk we spent time with Una (a pH student in the bat lab and an expert on Irish bats!)
We learned about the various 9 definite species of bat that are domiciled in Ireland and the ‘blow in’ species on a visit from the UK. It’s great to see a ‘cool’ use for the callipers, we are sick of measuring beakers and balls in school!
Calipers-more useful than you think!
Now I can tell the students that they are part of a zoologists ‘toolkit’ when identifying species based on morphology only. We actually measured the forearm of some preserved bats, identified the species using keys (way more cool than using them for Irish plants on the hockey field!)
We learned about the frequencies of echolocation and how to use the bat sensors to pick up the variety of species in one location. Really loved this talk, completely fascinating. The bat walk- totally amazing!!!!!!!!! If Santa is stumped for good gifts-a bat sensor would be a winner!!!!!!! Immediately we picked up a Soprano Pipistrelle- the first time I have actually seen the perfect outline of a bat flying over head!, there was no mistaking that it was a bat and not a sparrow. We also identified Leislers bat, the only elusive guy was Daubentens bat! Totally amazing- free and a great activity for holidays- buy a bat sensor!
Different species of bat-different echolocation frequency, one species cannot hear the other- did this change in echolocation frequencies result in speciation? When did it occur? Could all bats echo locate and then some lost that ability through genetic change?
Today was spent trying to fine tune the content of the student workbook-the most difficult task is to tone the science down to meet the understanding and stimulate interest in your Irish Teen 15 year old.
Much of today was also spent studying- you get interested in the lab work and the bioinformatics but I need to paint the full picture for myself. Professor Teeling gave Clare and I a tutorial on bat evolution on Wednesday and it sparked an interest.
You can’t mention bats without mentioning their key feature-echlocation. There are numerous studies ongoing which are looking at the how and why of this process-
- one area of research involving sympatric (same country) evolution-Basically you have many bats living in the same area-the frequency of their echolocation changes (possible gene mutation?) and this results in reproductive isolation- no genetic drift- this population of bats change genetically over time, they are unable to communicate with and reproduce with those bats using a different frequency-this group changes slowly overtime, so much that they only breed with bats using their frequency-a new species is born.
- Bats seem to form new species much faster than other mammals-this is why there is such interest in studying them-are they evolving faster? Why is this happening? What gene or genes are responsible?
- What would you discover genetically if you sequenced the Fox P2 gene in the DNA of different bat species known to have distinct echolocating frequencies? What would you see if you aligned the sequences? Would there be deletions, insertions etc, When you create the phylogenetic tree of maximum likelihood where would the nodes appear- compare the tree to fossil study and date the branches-what would you discover? If you date the branching or divergence back to period in history, was there any significant incident in the environment?
This is only some of the information that you get absorbed in when you are in the bat lab-It pushes you to study and try to get up to speed. The study aspect is fantastic- you can sit and read papers, read campbell, take notes etc., Bat Lab and all the cool people that make up the Bat Lab-INSPIRE!
DNA-the evolutionary pathway can truly only be mapped in similar species of mammals using DNA.
Bioinformatics-It’s a bit like nucleotide mining-Its the bases that you are interested in. When you align the sequences of similar bat species you should see gaps- insertions of bases, removal of bases- the fewer the gaps the more closely related they are. You can create a blueprint of change.
BLAST, CLUSTAL & MEGA-Serious computing power
Science meets Art-beautiful work outside the UCD science block!
We have spent the last 3 days learning about sequencing, alignment, creating trees, the algorithms and the role of probability in the program’s used to create the trees and to grade their reliability.
So basically maths is vital to the creation of the trees.
Last day with Graham and without any help we have to Search the database for a sequence of choice, BLAST the sequence and select organisms with listed that match somewhat our target.
MEGA- model analysis tool, MEGA is giving the best MODEL to create your tree with
We align the sequences using Clustal, import the alignment into MEGA(programme that will create the tree), select the model that will create the most mathematically reliable model and create the tree.
Phylogenetic Tree-Alpacas, Camels and Platypus
All managed thanks to Graham’s teaching skills!
We also checked out Chimera and viewed 3D model images of proteins we had BLASTED-Cytochrome b and opsin ( red and green colour receptor).
Back to bioinformatics- getting to know the software is the turning point- once Graham had re-taught Clare and I the steps involved in Blasting sequences and then aligning them- we felt we were getting somewhere.
First Phylogenetic Tree.
Graham is an absolute expert-he explains the role of probability and statistics in the running of MEGA and BLAST.
In a straight forward way this is how you begin to create a phylogenetic tree using DNA sequences from a bat gene:
1.DNA sample you purified is sent away for sequencing.
2. The sequence is now inputted into BLAST where it is compared against thousands of other sequences. The sequence is identified and linked to similar organisms- in this case there is a 100% match to a particular Myotis species of bat. Any other similarity to the same gene or sequence in other bat species is also indicated.
3. You want to create a phylogenetic tree- a diagram which shows the evolutionary pathway-was this sequence once the same in a common ancestor and is it possible to trace back a stage or branch at which it evolved or changed.
4. Select the search results listed in BLAST and use Clustal X to align the sequences- you are using a programme to position the sequences from each of the species of bat to gain the best fit or match possible. Too large a task without Clustal X.
5. Load the alignment file into MEGA- this will then use the aligned sequences to create a phylogenetic tree- various tools to analyse the reliability of your tree are used in MEGA- this is known as boot-strapping.
6. A tree is produced with numbers at each Node- over 50% the connection is somewhat likely but really 80% upwards is sought.
So with much ado we created our first phylogenetic trees.
Yesterday Graham introduced Clare and I to BLAST, we learned how to use BLAST to identify sequences and in species identification. This is only step 1.
Species identified and similarities with other species identified but you now must use the sequence and in the Bat lab the production of a phylogenetic tree is the goal.
Phylogenetic trees (gene trees which should an evolutionary pathway) are constructed using Homologs ( the sequences must have descended from a common ancestor) BLAST helps you pick sequences that could be used in the tree construction. Each sequence that appears when you do a BLAST analysis will have an ‘e’ value- values of less than e -5 are accepted as Homologs and can be used in alignment.
What??????? I know seriously complicated and all based on algorithms!
Selected the Homologs and input them into a program that aligns them, it places them underneath each other trying to match the sequences with best fit, creating spaces to achieve this fit.
This is all done on the computer using free tool such as MEGA but mainly Clustal X. This is the beginning of the tree creating process.
Did you know that Clustal X is one of the most widely cited scientific publications in the history of biology. Des Higgins in UCD one of its creators is one of the most cited computer scientists in the world!
Intense morning with slow laptops and programmes (MEGA) slowing as the scientists on the other side of the planet log on. Learned a lot and trying to study theory to help paint the picture.
Bioinformatics with Graham-Genetic research seems to go hand in hand with bioinformatics. A great deal of energy and time goes into the extraction, purification and amplification of DNA, then what? You produce this beautiful DNA barcode but unless you sequence the DNA and use the information, you are limited in what you can say about your DNA.
The bat lab uses DNA (mitochondrial DNA) for identification of species (only 1 use of many)
You extracted DNA from a number of bats of unknown species, you have isolated cytochrome b, a gene which used in linking closely related members. PCR amplified a particular sequence in the gene for which primers were specifically selected. After electrophoresis satisfied you that PCR worked you then quantify the amount of DNA, use the Nano Drop.
The samples if they contain enough DNA, are then sent to an outside sequencing company which come up with A,T,G,C’s . This is is where bioinformatics comes in. Databases such as BLAST and NCBI contain thousands of fully sequenced genes, you can enter your sequence and compare it with those already inputted and it will match up to all homologes (the same gene in different species) and this is used for further analysis.
These data bases match per nucleotide and if your sequence matches up perfectly with that of one in the database it will be very clear.
Perfect match with 100% of nucleotides
Look for the mitochondria-Mitichondrial DNA is most often used in evolutionary genetics- Cytochrome b is the gene we are working with.
Friday has arrived-Last day of Gel electrophoresis before we embark on bioinformatics.
Samples that underwent PCR last night are now ready to be visualised.
You by now know the routine-prepare the gel- let it set. Get your samples, the positive and negative controls and the DNA ladder. Add 2ul of the UV safe dye into your wells and then add the volume of sample you have decided on to the wells with the dye.
Place the gel in the buffer and load the lanes of the gel carefully. Run the electricity for 20 mins. Remove gel and place in the UV chamber and run the programme. DNA barcode is visualised on the screen.
Unfortunately our winning streak abandoned us this morning- quality was not great and so we decided to repeat the electrophoresis again. Just waiting now for the gel to set, will let you know how we get on.
1% Agarose Gel Left to set.
Afternoon DNA barcode looks great. Electrophoresis worked really well.
DNA band very visible in third lane from the left. Nothing in the negative control which is very important. Clare’s results are in top lanes, mine are in the bottom lanes.
First week ends on a high, it’s amazing how much I have learned in terms of lab work and genetic theory. Thanks to all at the Bat lab and special thanks to Dr.Lao.