Bacteriophage Evolution Lab – Week 5
10-9-06
Introduction
The sequencing reactions you will set up today will take a only a small part of the
lab time. The majority of the time will be used to discuss the article on influenza and
your course of action for the rest of the phage evolution labs.
Our goal in these phage evolution labs has been to introduce you to some of the
lab techniques, some of the ways of thinking, and some of the ways of communicating
when doing genetic research. Until now, you have been involved primarily in the lab
techniques. During this lab and the next lab you will have the chance to think in more
depth about the biology and evolution of phage. This will give you the chance to think
more critically about what has or hasn’t occurred during these evolutions, and how you
might interprete the sequence changes you may see. It will also give you a chance to
consider the limitations of these experiments, possible conclusions you might draw from
them, and further questions you may raise as a result of your conclusions. These
conclusions and questions will become the basis for the research paper you write and the
basis for further experiments you will propose if you were going to continue with this
research.
As you think ahead toward your research paper you may find it interesting and
useful to view what is occurring with the phage and the E. coli from different
perspectives.
Think about the molecular biology that is occurring: The initial binding of the
phage to the E. coli, the injection of DNA into the bacterial cell, the transcription of
specific genes and replication of the DNA, interactions between the molecular machinery
of the cell and of the phage, assembly of the phage procapsid, introduction of DNA into
the phage procapsids, and release of the final mature phage capsid which contains the
phage DNA. There are times when it is useful to think about the whole infection process
or specific steps of the infection process. There are also times when it is useful to think
about the process at the level of specific, small, molecular regions (a promoter, a single
protein’s structure, the amino acids within that protein).
Alternatively, you might think about the evolution that is occurring and the
population that is being subjected to evolution. Can study of evolutionary change help us
to better understand the biology of organisms? What are some of the major variables
affecting evolution? Mutation rate? Population size? Strength of selection? These are
variables that will be covered in more detail near the end of the course, but ones that will
still be useful to think about now.
Think about evolution from a detailed perspective and then from a much broader
perspective. What kinds of sequence changes will improve the fitness of these phage
when they are grown at low temperature? Are there multiple routes for the initial
ancestor, ID8, to follow in order to achieve greater fitness? What questions do these
thoughts, and possibly the data you get out of these experiments, raise?
How can study of small amounts of evolutionary change (microevolution)
improve our understanding of large amounts of evolutionary change (macroevolution)?
Does study of macroevolution help frame questions to be asked by study of
1
microevolution? How can different organisms (model systems) be used to address
questions about evolutionary change?
At the end of this write-up are some pages of the genetics text that contain
information you may find useful. Some useful web sites and figures are also included.
Materials Needed/group
1. Gloves to wear when you are handling materials
2. Cleaned up PCR reactions from last week and a rack to hold them (Keep tubes on ice
when not using them.)
3. Four PCR tubes to set up the sequencing reactions in and a small rack for those tubes
4. p10 micropipettors and tips
5. A waste container for used tips
6. A tube of Big Dye (keep on ice except when actually using. It’s a bit sensitive to light
and is extremely expensive stuff!) The Big Dye contains buffer, heat stable polymerase,
dNTP’s (dATP, dCTP, dGTP, dTTP), ddNTP’s (ddATP, ddCTP, ddGTP, ddTTP), and
Mg++.
7. A tube of H2O
8. Two tubes of sequencing primers to be used with your “A” PCR product:
UG1447A, labeled “14A”
UG1997A, labeled “19A”
9. Two tubes of sequencing primers to be used with your “B” PCR product:
UG2495B, labeled “24B”
UG3162B, labeled “31B”
Determination of Concentration of PCR Products
Use the gel photos from last week’s lab to determine whether your reactions worked as
you predicted they would. If they did, determine the concentration of each PCR band. If
not, talk to your TA about using some of the PCR product from another group. If you
need to do this BE SURE to get the information from them about the source of the phage
they used. Each of you should write this information down. You need to keep track of
the initial phage suspension that was used for all reactions.
1. Each of you can get a gel photo from the TA, showing your products (DNA bands)
and DNA mass ladders with known amounts of DNA in each band.
2. Compare the intensity of your bands to the intensity of the known bands to estimate
the amount of DNA that was in the 2ul that you loaded on the gel. Note: you actually
loaded a volume of 10ul into each gel lane last week, but that 10ul only contained 2ul of
your cleaned up PCR reaction.
3. Determine what volume of each of your cleaned up PCR samples needs to be added to
the sequencing reaction to give you 10-25 ng of DNA in each reaction. Check your
concentration estimates with your TA. If you need to add more than 4 ul of your sample,
or less than 0.8ul, discuss this with your TA.
2
Setting up Sequencing Reactions
Before setting up your reactions, look at the map of ID8 below. Use the
information from last week to draw in the region covered by PCR product A and product
B. Draw in the location of each sequencing primer. Include arrows to show which
direction sequencing will extend for each primer. Which gene is each primer
sequencing?
1. You will be setting up four sequencing reactions—two with each DNA template.
Wipe down your bench area and wear gloves.
1. Label the four PCR tubes on their sides with your group’s initials and: 14A, 19A, 24B,
and 31B. The TA will eventually assign you numbers to label the top of each of your
reaction tubes. After you have set up the reactions, you can close the tubes and write
those four numbers on the top, flat part of the lids where they won’t wear off.
2. Each sequencing reaction will contain the following components:
5ul of one primer
10-25 ng of your DNA template
1ul of Big Dye
H2O to give a total volume of 10ul
3. Given the amount of each template that you need to add, figure out how much water
you need to add to each reaction in order to have a final volume of 10ul.
4. Be sure to wear gloves when you add the components to each tube (PCR tubes can be
at room temperature while setting up reactions):
Tube
14A
19A
24B
31B
Primer
5ul 14A
5ul 19A
5ul 24B
5ul 31B
DNA
___ul A
___ul A
___ul B
___ul B
Big Dye
1ul
1ul
1ul
1ul
H2O Class Number
___ul
____
___ul
____
___ul
____
___ul
____
5. Cap the tubes, label the tops and write the labeling above. Put them on ice or where
your TA wants them to go. Put things away. Save your unused PCR products.
Post-lab sequencing reactions, clean-up, and running on sequencer
Cycle sequencing will be done on the reactions. This will be done in the same
PCR machines that were used last week, with very similar cycling profiles but with
slightly different annealing temperatures that are optimized for the primers you have used
and shorter elongation times because the maximum sized products will only be about
700-800 bases. Note that since only one primer is used in each reaction, amplification
will be arithmetic rather than exponential and the products will be single stranded DNA.
3
Samples will be cleaned up after cycling to remove excess nucleotides and primers, and
Ursel will then run them on the automated DNA sequencer.
At the beginning of the bioinformatics lab next week you will be able to
download and work with your four sequencing files. If you find sequence changes, you
will be able to map those onto 3-D structures of phage procapsids and capsids.
5000
H
1000
A
A*
G
ID8
5540 bp
B
K
4000
C
E
D
F
2000
J
3000
Chapter 23 of the text is devoted to population genetics and evolution. If you have
time, a reading of this chapter will give you a better perspective for viewing these phage
evolution experiments and experimental evolution in general. Pages 676, 677, and 697 to
the end of the chapter may be especially helpful
A look at the web sites listed below before the lab next week will probably help you better
understand the 3-D protein images you will be manipulating in that lab.
The RCSB Protein Data Bank is “An Information Portal to Biological Macromolecular
Structures.” Their “molecule of the month” (major nerds, huh!) about a year ago was the
phiX174 capsid. Check it out:
http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb2_
2.html
4
Web site for Dr. Ben Fane’s lab, a lab that does research on the assembly of phiX174 and
other viruses from their proteins and nucleic acids:
http://microvet.arizona.edu/Faculty/fane/fane.html
The research address from Fane’s lab’s web site:
http://microvet.arizona.edu/research/fane/fane.html
Their introduction to assembly of viruses like phiX174 and ID8:
The proper assembly of proteins and nucleic acids into biologically active virions
involves numerous and diverse macromolecular interactions. While structural proteins must
correctly interact, proper morphogenesis is equally dependent on scaffolding proteins,
which are transiently associated with nascent protein complexes during virion assembly.
Scaffolding proteins can also mediate conformational switches: structural changes that
cause the next assembly process to differ from the previous one. With developed genetics
and biochemistry, the Microviridae system (øX174, G4 and alpha3) is well suited for the
proposed research. At least six assembly intermediates can be purified. The viral assembly
pathway is depicted in Figure 1. The atomic structures of the øX174 virion and procapsid,
containing two scaffolding proteins, have been solved (McKenna et al., 1992, 1994;
Dokland et al., 1997, 1999) . Hence, genetic and biochemical data can be interpreted within
a structural context.
Figure 1: Microviridae Morphogenesis:
5
Check out the six assembly intermediates shown in Figure 1 above. The D, F, B, G, H, and
J proteins in Figure 1 all originate from the corresponding genes in the PhiX174 map,
which is very similar to the ID8 map.
A protein structure refresher in three figures!
6