Masterclass 7 July 2008

Biology and Technology

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General Introduction

Welcome to the Masterclass – Biology and Technology. This

information pack includes introductions to the people who will be working with you today and summaries of the activities you will

participate in. It also contains important information about how to

keep safe during today’s activities.

Timetable

Table of Contents

PEOPLE INVOLVED 3

SAFETY AND RESPONSIBILITY 4

EVOLUTION AND CLASSIFICATION 6

MODELING BIODIVERSITY 14

ELECTRON MICROSCOPY AND NANOSCIENCE 21

FURTHER INFORMATION AND LINKS 24

Time Activity Location

11.00-11.10 Arrival/Housekeeping/Agenda/coffee Maths and

Physics Foyer

11.10-11.30 Mini lecture by Dr. Tom Reader Maths and

Physics A1

11.35-12.45 Evolution and Classification Pope A26

12.50-1.30 Lunch and info on courses Maths and

Physics Foyer

1.35-2.45 Modeling Biodiversity Pope A26

2.50-3.50 Electron microscopy with Dr. Michael Fay NNNC

3.50-4pm Question and Answer Session Maths and

Physics A1

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People involved

Dr. Tom Reader is a lecturer in ecology. He studied for his BSc at Lancaster University, completed his PhD at Cambridge University,

and then worked at the University of Sydney before coming to the

University of Nottingham. He studies population and community

ecology and animal behaviour. He has always been keen to

communicate his research to a wider audience, and his outreach

experiences have included TV and radio appearances, school visits

and contributions to various public events.

Dr. Michael Fay is responsible for the electron microscope suite at

the NNNC. He studied for his BSc at Leicester University, an MSc at Warwick University, and completed his PhD at Sheffield University

before coming to the University of Nottingham in 2000. He originally

worked on electron microscopy of semiconductors, but now looks at a

wide range of materials from spider silk to pharmaceuticals to carbon nanotubes.

Pamela Styles is originally from Birmingham, where she completed

A levels in Biology, Chemistry, Maths, General Studies and an AS

level in French. She completed a degree in Genetics at the

University of Nottingham, and decided to stay to do a PhD in

Genetics. She is now in the second year of a computer-based PhD

research project on "the Evolution of Mobile DNA".

Tim Newbold is a PhD student in conservation ecology. He

completed his undergraduate degree in Zoology at Nottingham, before travelling to Egypt to study its biodiversity for a year. He then

returned to Nottingham to begin his PhD, continuing to work on the

biodiversity of Egypt.

Rohanna Dow is a PhD student in Biology. She completed A-levels

in Biology, Chemistry and English and an AS level in Physics before studying for a BSc in Zoology at the University of Nottingham. After

completing her degree, she took two years out to travel and work.

She is now working on a PhD investigating mimicry within natural

and computer generated habitats.

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Safety and Responsibility

We want to make sure your visit to the University of Nottingham is

as safe and interesting as possible. Please read the notes below and, while on campus today, please ensure you follow all instructions from

your teachers and the researchers leading the activities.

Please be aware of where you and your belongings are at all times.

In every building you visit, you will be told where the nearest emergency exit is. If you hear an alarm, please make your way

quickly and calmly to the exit.

No bags or coats should be taken into laboratories. In laboratories, please do not touch any equipment unless you are specifically invited

to do so.

When using computers on campus, you have a responsibility to act in

accordance with the University’s Code of Practice (next page).

Please read this. If you do not agree to the Code of Practice, you

must not use the computers at the University of Nottingham.

If at any point in the day you are injured, you must inform the researcher you are working with or another member of staff

immediately. Certified First Aiders will be available to help you.

Thank you very much. With your cooperation we can have a safe

and productive day. A full risk assessment has been completed for

today’s activities. If you would like to see a copy, please ask.

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IT

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Evolution and Classification

In this section you will:

� Learn how to make an evolutionary tree of species by sequence

searching, extraction and running the tree-building programs

to investigate how species are related.

� Explore why classification of organisms is important, and how

humans are classified. Identify species and relationships by

examining skulls and bones

Using Computers to Make Evolutionary Trees

1. Double click on the Novell Application Launcher (NAL) icon on

the desktop.

2. In the left hand pane, click on School and Departmental ���� Biology ���� Fundamentals of Molecular Evolution. Three icons

will appear in the right hand pane: ClustalX, NJ Plot and Protpars

(Figure 1). We will be using ClustalX and NJ Plot today. You can minimise NAL for now, but keep it open.

Figure 1

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3. Open an Internet Explorer browser window by double clicking on

the Internet Explorer icon on the desktop.

4. Type in the following address:

http://www.pubmed.com

This will take you to the National Centre for Biotechnology

Information (NCBI) website. Through this website, you can access

most of the publicly available DNA and protein sequence data. This is

freely accessible to anyone.

5. On the bar along the top, select “Protein” from the drop down list

next to Search (Figure 2).

6. If you are making the bacteria tree, you will be using the S12 protein, which is a component of the 30S subunit of the bacterial

ribosome. To locate this protein, try searching with something like

“Bacillus anthracis S12 30S”.

Step 6: Type the protein you

want to search for in this box

Step 5:

Select Protein

Step 6: Then

click Go to

search for your

protein

Figure 2

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For the mammal tree, you will be using the amelogenin protein.

This protein is a major component of tooth enamel. Type in the name of the species and the protein name, e.g. “dog amelogenin”, or

“Canis familiaris amelogenin”. Then click Go (Figure 2).

7. A list of search results will appear. The one you want should be

near the top! The name of the protein and the name of the species

will usually appear. When you’ve found the protein you want, click on

the reference number in blue (Figure 3).

8. Details about this protein will come up, such as what species it’s

found in and the names of the authors who submitted the sequence

(Figure 4).

This format is called GenPept format, and contains more information

than we need to make a tree. The sequence format is shown in the

top left (GenPept). Click on the arrow and select “FASTA” from the

drop down list (Figure 4). Fasta is a much simpler file format. It

consists of a “>” symbol, followed by a description of the sequence. Underneath this descriptive line is the amino acid sequence, e.g.

Step 7: Click on the

blue reference number

to open up the

database record for this

protein

Figure 3

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>Human Amelogenin MGTWILFACLLGAAFAMPLPPHPGHPGYINFSYEVLTPLKWYQSIRPPYPSYGYEPMG

GWLHHQIIPVLSQQHPPTHTLQPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQHHQ

PNLPPPAQQPYQPQPVQPQPHQPMQPQPPVHPMQPLPPQPPLPPMFPMQPLPPMLPDL

TLEAWPSTDKTKREEVD

9. The sequence will appear (Figure 5), and in most cases will begin

with “M” (methionine). All protein sequences start with methionine.

Where the sequence in the database starts with a different amino

acid, this might mean that part of the protein is missing. Clustal will

introduce a gap to account for this if it is the case. Select the amino

acid sequence and copy it.

Unique database

reference number

Species

Classification of the

species

Length of protein

(117 amino acids)

Some information

about the animal

the sequence came

from

Name of the protein

What the protein does

Step 8: Select

FASTA from this list

Figure 4

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The Fasta descriptive line produced by the database is usually long

and complicated, so don’t copy it over. Just copy the sequence. You will be able to add your own name for the sequence.

10. Open Notepad (Start � Programs � Accessories � Notepad).

Paste in your amino acid sequence. On the line above it, type “>”

and then give your sequence a title. This title is what will appear on

the tip in your tree, so it is best to give it the name of the species the

sequence came from, e.g. “Dog”, or the disease caused by the bacterium it comes from, e.g. “Anthrax” (Figure 6).

11. Return to the Internet Explorer browser window. Click the back button until you return to the screen shown in Figure 7.

Now repeat stages 6-10 to search for, copy, and paste into your

notepad file, the other sequences you need for your tree.

The FASTA

descriptive line

FASTA

sequences

always begin

with a > symbol

Amino acid sequence

Figure 5

Figure 6

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12. When you have all six sequences in a notepad file, with no spaces between them (Figure 8), you are ready to make the tree.

Save the file, for example, “amelogenin.txt”, “mammals.txt” or

“bacteria.txt”. Save it in the My Documents folder.

13. Return to the NAL window (Figure 1). Double click on the ClustalX icon. This opens the ClustalX alignment program. Go to File ���� Load sequences, and then browse to find your notepad file

with the sequences in it. Click on it, then select “Open”. Your sequences should appear in the ClustalX window.

Figure 7

Figure 8

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14. Now you need to align your sequences. Go to Alignment ���� Do

complete alignment. A box will pop up. Click on “Align”. You will notice that dashes (-) are introduced where the program believes

insertions and deletions have occurred. Asterisks (*) along the top of

the sequences indicate positions which have the same amino acid in

all six species.

15. Now that the sequences are aligned, you are ready to make a

tree. ClustalX has another program incorporated into it, called

Neighbour Joining (N-J). This is a “phylogenetic reconstruction”,

or tree-building program. Go to Trees and click on “Correct for multiple substitutions”. Click on Trees again, and select “Bootstrap N-J Tree” (Figure 9).

16. A window will appear asking how many bootstrap replicates you

want, and what to call the output file (Figure 10). The default values are fine, so just click on OK. NJ will do 1000 bootstrap replicates by

default.

Figure 9

Figure 10

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17. When your tree is ready, a message will appear in the bottom left

of the ClustalX window to say your tree has been created. You can now return to NAL, and open NJ Plot. This will allow you to view

your tree.

18. In NJ plot, go to File ���� Open. Then find the tree file that N-J

has created. It will end in the file extension “.phb”, so if you called your original file “mammals.txt”, your tree will be called

“mammals.phb”.

19. Select your phb file and click open. Your tree will appear. This

tree is unrooted, and you need to let NJ Plot know which species to

use as outgroup for it to draw the tree correctly. Click on “New outgroup” at the top, and hashes (#) will appear next to the species’ names. Click on the hash # next to the species you wish to

use as outgroup (Pyrococcus furiosus or Opossum). Then click on

“Show tree”.

20. You can also click on “Bootstrap values”. The number of

bootstrap replicates (out of 1000) which support that branch will be shown above the branch. Anything over 50% (500) isn’t bad, over

70% (700) is very good.

Questions

Bacteria: What kind of bacterium do you think plague evolved from?

----------------------------------------------------------------------------

Mammals: What are humans’ closest relatives outside of the

primates?

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Modeling Biodiversity

In this section, you will:

� Discuss how we might measure biodiversity and how we might

use this knowledge to decide which areas of the world most

need conserving.

� Consider what determines where a species is found and how

we can model this using computers.

� Create your own species distribution model using specialist

software called Maxent.

Biodiversity

Biodiversity is a term used to describe the variety of life on Earth.

Conservationists generally agree that we should try to conserve as

much biodiversity as possible given the resources available. But how

might we measure biodiversity?

A common measure of biodiversity is species richness. This is simply

the total number of species found in a particular place and is

relatively easy to calculate, making it useful for deciding which areas

to save. Working with the person sitting next to you, try to answer the following questions. We will discuss them together shortly:

1. Some habitats have more species than others. Try to list some

habitats that have lots of species and some that have few.

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2. Can you think of any problems with using species richness to

decide which areas need to be conserved? Will all species be conserved equally well?

If scientists have been to a place, they can estimate the number of

species found there. However, many areas have never been visited,

often because they are very difficult to get to. We need some way of predicting the diversity of unknown sites from what we know about

the places that we have already visited. To do this, we need to

understand what affects where species live.

Species Distributions

Some species live in tropical rainforests, some in the Arctic, while

others live in the desert. What determines where a species is found?

Working in pairs again, list as many things as you can think of that

might affect whether a species can live in an area or not.

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Modelling Species Distributions

Computer models attempt to capture and explain the important

aspects of real-world situations. In the case of species distributions

we attempt to find what determines the distribution of a species so

that we can predict whether the species will occur in places that have

never been visited before.

Computer models almost always simplify the real world, especially

when they are trying to explain biological processes. Where a species

is found may be determined by many factors (some of which we discussed previously). All we can hope to do is use as many factors

as possible. This means that the model predicts where a species

could be found, not where it is found.

Species distribution models take a list of sites where a given species

has been recorded. Information on climate, habitat and other

environmental factors (usually worked out from satellite images) at

these sites is used to build up a picture of the environmental

conditions that the species lives in. This is then applied to the

environmental conditions of unknown areas to predict whether the

species could live there.

Building Your Own Species Distribution Model

All the files that you need for the practical today can be found in the

C:\Masterclass folder on your computer.

In there you will find some picture files (the file type is jpeg). These show maps of sites where species have been seen and also maps of

some environmental variables (climate and habitat). Have a look at

these and see if you can spot any patterns in the environmental

conditions of the places that species are living in.

Now to build your model. The software that you need is also in the

C:\Masterclass folder.

1. Double click on the file called Maxent (there are 2 files with the same name – you need to click on the one that has the

file type ‘MS-DOS Batch File’). You should get a window like

the one shown below:

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2. Next choose one of the four species to model. The sites that

these species have been seen in are listed in the Excel files

(Red Fox, Lesser Gerbil, Dorcas Gazelle and Jungle Cat) with

file type ‘Microsoft Office Excel Comma Separated Values

File’.

3. You need to load the samples that you have chosen into the

Maxent software. Click on the ‘Browse’ button next to the

‘Samples’ box, navigate to the C:\Masterclass folder and

double-click on the file for your chosen species.

4. Next you need to load the environmental variables. These

are found in the ‘Variables’ folder within the C:\Masterclass

folder. Click ‘Browse’ next to the ‘Environmental Layers’ box,

navigate to the C:\Masterclass folder, click once on

‘Variables’, then click ‘Open’.

5. The habitats variable is divided into categories, so in the list

of environmental variables you need to change the box next

to habitats so that it says ‘categorical’. 6. Click on the boxes next to ‘Create response curves’ and

‘Make pictures of predictions’ so that the boxes are checked.

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7. Click on the ‘Browse’ button next to the ‘Output directory’

box, navigate to the C:\Masterclass folder again, click once on the folder called ‘Model’ and click open. Your model will be

saved in this directory.

8. Finally, click ‘Run’ and your model will begin.

9. When the boxes showing model progress (as shown below) have disappeared, your model is finished. This should only

take a couple of minutes.

10. If you get a warning message that says ‘Sample at …………. in ………….. is out of bounds, skipping’ then click ‘Suppress

similar warnings’.

11. If you get a warning message that says ‘Sample at ……….. in

species.csv is missing some environmental data’ then click

‘Ignore such samples (safer)’.

12. Leave the Maxent software window and return to the

C:\Masterclass folder. Then double-click on the folder called

Model.

13. There should be an internet file with the same name as the

species you just modelled (e.g. Jungle_cat, Lesser_gerbil

etc.). Double-click on this file.

14. If you scroll down, you should be able to see a map of the

predicted distribution, where the environment is suitable for your chosen species. Warm colours (red and orange) show

very suitable places. Cool colours (blue and green) show less

suitable places. The white dots show places where the

species has been seen (the places that you used to make your model).

15. If you scroll down further you will see graphs showing the

relationship between where your species was seen and the

environmental variables.

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What Can We Use the Models For?

Working in pairs again, have a think about what the models tell us

about the species. What does your model show?

How could we use the models?

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How do you think that we could improve the predictions?

If you like, you can e-mail the map to yourself. It is a PNG file with

the same name as your species and is found in

‘C:\Masterclass\Model\plots

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Electron Microscopy and Nanoscience

In this section, you will:

� Visit the Nottingham Nanotechnology and Nanoscience Centre

(NNNC)

� Hear a brief lecture about electron microscopy and nanoscience and view nano-scale images taken at the centre.

� Get an in-depth look at state of the art scanning and

transmission electron microscopes (SEM and TEM) and see

them in action.

� Tour the NNNC, including the atomic force spectrometry suite

and the PEEM lab (proton emitting electron microscope).

What is Nanoscience?

“Nanoscience and nanotechnology involve studying and working with

matter on an ultra-small scale. One nanometre is one-millionth of a

millimetre and a single human hair is around 80,000 nanometres in width. Nanoscience and nanotechnology encompass a range of

techniques rather than a single discipline, and stretch across the

whole spectrum of science, touching medicine, physics, engineering

and chemistry.

Nanomaterials can be natural or manmade. For example,

nanoparticles are produced naturally by plants, algae and volcanic

activity. They have also been created for thousands of years as

products of cooking and burning, and more recently from vehicle

exhausts. Some proteins in the body, which control things like flexing

muscles and repairing cells, are nanosized. We can set out to make

nanomaterials in a variety of different ways. Some nanomaterials can

assemble themselves from their components. Carbon fragments, for

example, can self-assemble into nanotubes in this way. Another

approach, used in the production of computer chips, is to etch

nanomaterials from larger pieces of material. Increasingly, these two

methods are converging, leading to exciting new production

techniques.

Powerful microscopes have been developed which allow

researchers not only to look more closely at atoms and molecules,

but also to pick them up and move them around to form basic

nanostructures. This allows some nanomaterials to be built molecule by molecule. For instance, this technique was used to manipulate

atoms to spell out the IBM logo. However at present this technique is

extremely laborious and unsuitable as an industrial process.”

- The Royal Society

http://royalsociety.org/page.asp?id=1211

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Electron Microscopy

SEM operated by Dr Hannah Edwards

The FEI Quanta200 3D DualBeam FIB/SEM uses a focussed ion

beam (FIB) and scanning electron microscope (SEM) to image,

analyse and process samples. Both the ion beam and the electron

beam can be scanned across the sample with information (such as

scattered electrons or X-rays) measured for each point and plotted

on the computer screen. The ion beams, which have a resolution

down to 10nm, can be used to very accurately sputter material from

the sample. This means that cross-sections can be made at specific features of interest and analysed using the SEM, or by making a

sample with a very thin (~100nm) region, prepared for analysis

using the transmission electron microscope. This technique is

particularly useful for samples which contain materials of very

different hardness, such as biological cells on metals or ceramics.

The FIB/SEM is equipped with a Quoruom Technologies PP2000T

Cryo transfer system, which allows frozen samples to be prepared in a similar manner. This means that biological samples, food, oils or

even liquids can be frozen to -140 degrees C, cross-sectioned and

analysed.

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TEM operated by Dr Mike Fay

The JEOL 2100F Transmission electron microscope (TEM) has a point resolution down to 0.19nm, which means that individual large

atoms can be seen. Using very thin (~100nm) samples, the 200 kV

electron beam passes through the sample to produce images that

can be recorded on a specialised high-resolution digital camera. The

microscope also has a spectrometer than measures the energy of the

electrons, and Scanning Transmission Electron Microscopy (STEM),

which combines the abilities of SEM and TEM in one instrument. By

measuring X-rays emitted from the sample, or the energy of

electrons after they have passed through the sample, the elemental

composition of samples can be measured at scales down to 1 nm resolution. Special holders allow samples to be frozen with liquid

nitrogen or heated up to 1000 degrees C in the column, reacted with

gases or even analysed with a scanning tunnelling microscope tip

within the electron microscope.

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Further Information and Links Thank you for coming to the Masterclass! We hope you enjoyed the

day!

For further information on anything you have seen today or details

about studying at the University of Nottingham, please email

scienceoutreachproject@nottingham.ac.uk, or go to the project

website at www.nottingham.ac.uk/sop.

For additional information about any of the departments involved in the Science Outreach Project or today’s event, check out the

following webpages:

School of Biology: www.nottingham.ac.uk/biology

School of Pharmacy: www.nottingham.ac.uk/pharmacy

School of Chemistry: www.nottingham.ac.uk/chemistry

Faculty of Engineering: www.engineering.nottingham.ac.uk

NNNC: http://www.nottingham.ac.uk/nano/