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Lab handout one
Created by AlexiaLamb
General
Author: AlexiaLamb
Laboratory:
Date: 1/31/17
Abstract
Forward and reverse genetics
Analysis of mutants is the main tool used by geneticists to understand how the information
in DNA is translated into the phenotype. These analyses can be broadly grouped into two
categories, forward genetics and reverse genetics.
Forward genetic analysis is the older of the two methods, but is still a standard and modern
way of determining the genetic basis of a trait. In brief, the procedure for analyzing a trait
using forward genetics is to (1) start with an interest in a phenotype (trait), (2) find
individual organisms that vary in that trait, or create mutants that vary for that trait, (3)
analyze the genomes of those individuals to see what genetic variation is present (3) test
whether that variation correlates with the variation in the phenotype, and then (4) perform
functional testing to confirm that the candidate gene you have identified via steps 1-3 has
the function that you hypothesized. Functional testing can include biochemical analyses,
additional mutation, allele substitution, and complementation testing.
Reverse genetic analysis has grown out of the increasing availability of genome sequences
and precise mutagenesis techniques in some organisms, even down to the level of knocking
out genes in specific tissues or cells. Reverse genetic analyses start with a specific gene that
has been identified by sequence, for which we do not know the function. This identification
is often done by gene prediction software available independently or with genome
browsers, like Galaxy (https://usegalaxy.org/ ). We then determine the function of the
gene by knocking it out and looking for phenotypes that are no longer “wild type.” Once we
identify a trait that is affected by that gene, genetic studies can branch off in many
directions by analyzing the biochemical functions of encoded proteins, determining the
differences in tissue and/or temporal expression patterns, assessing the amount of natural
variation within or between species, etc.
In lab this semester we will be conducting a forward genetic screen to determine the
genetic basis of lactose metabolism in E. coli.
The genetics of lactose metabolism in E. coli is known, so we probably won’t be making any
publishable, ground-breaking discoveries in lab this semester. However, the process that
we will follow in lab is one that you may follow for any species, and for any trait in which
you are interested. Making and screening a mutant library for multicellular organisms
typically takes a long time – longer than we have this semester, but by using fast-growing
bacteria and a familiar phenotype, we can not only create the library, but also do the
follow-up screening, complementation testing, and sequencing that are necessary for
confirming candidate genes – all before the end of the semester.
The Lac operon is one of the most intensively studied genetic systems in the world. We will
discuss prokaryotic gene regulation in general, and the lac operon specifically, but it would
be helpful to do some background reading and look at the figure illustrating the Lac operon
in a basic genetics textbook, or online.
Our goal is to be able to start with a trait (lactose metabolism), use mutation to generate
individuals that are defective for the wild type version of our trait (unable to metabolize
lactose), find what gene is responsible for the trait (the one that was mutated in our mutant
strain), determine the sequence of that mutant gene, and propose one or more hypotheses
for why this mutation leads to a mutant phenotype.
The mutant hunt
When a geneticist embarks on a mutant hunt, one of the first things they need to do is
understand the phenotype they are investigating. In class, we have talked about
phenotypes as being clearly distinct, but there may be substantial variation in a phenotype
– and this is important to appreciate in order to define a mutant phenotype. For example, if
our trait of interest was purple flower color in pea plant blooms, how would we categorize
a lighter purple color? Would that variation likely be due to mutation, or is it likely an
environmental effect, or even random variation in color within the same color genotype
(variable penetrance, chance developmental errors, etc.)? As a geneticist, you need to be
aware of what the wild-type phenotype is and what constitutes a meaningful deviation
from that phenotype. In our mutant hunt, we are hoping to generate and identify mutant
strains that cannot metabolize lactose.
One important part of the mutant hunt is to define assay conditions that allow for rapid
phenotype screening and identification of mutant genotypes, which may be present at an
extremely low frequency.
When looking for mutants it would be ideal to establish conditions such that your mutants
are the only members of the population that are observable. A classic example of this is
selecting for antibiotic resistance on growth medium containing the antibiotic — only the
resistant mutants will survive. This type of mutant identification is known as
selection. Unfortunately, we are looking for a loss-of-function mutation, and both the
mutant and the wild type organisms are capable of growth.
As you can imagine, it is hard to visualize the absence of something (the absence of lactose
metabolism). Fortunately, one easy way to differentiate between different types of
bacterial cells is based on their differential metabolic properties. For example, some cells
may be able to utilize a particular carbon source, such as lactose, or produce a particular
metabolic intermediate. In these cases, we can often grow the potential mutants on a
specific food source, and with careful observation and a good understanding of the normal
range of bacterial colony shape, size, and color, we should be able to identify mutants. This
approach is known as a mutant screen. We will be using MacConkey agar plates, which are
described below, to visualize our phenotype.
From ASMscience.org http://asmscience.org/content/education/protocol/protocol.2855 :
MacConkey agar is a selective and differential media used for the isolation and
differentiation of non-fastidious gram-negative rods, particularly members of the family
Enterobacteriaceae and the genus Pseudomonas. The inclusion of crystal violet and bile
salts in the media prevent the growth of gram-positive bacteria and fastidious gramnegative bacteria, such as Neisseria and Pasteurella. The tolerance of gram-negative enteric
bacteria to bile is partly a result of the relatively bile-resistant outer membrane, which
hides the bile-sensitive cytoplasmic membrane (Nikaido, 1996). Other species specific bileresistance mechanisms have also been identified (Provenzano, et al. 2000; Thanassi et al.
1997).
Gram-negative bacteria growing on the media are differentiated by their ability to ferment
the sugar lactose. Bacteria that ferment lactose cause the pH of the media to drop and the
resultant change in pH is detected by neutral red, which is red in color at pH's below 6.8. As
the pH drops, neutral red is absorbed by the bacteria, which appear as bright pink to red
colonies on the agar.
The color of the medium surrounding Gram negative bacteria may also change. Strongly
lactose fermenting bacteria produce sufficient acid to cause precipitation of the bile salts,
resulting in a pink halo in the medium surrounding individual colonies or areas of
confluent growth. Bacteria with weaker lactose fermentation growing on MacConkey agar
will still appear pink to red but will not be surrounded by a pink halo in the surrounding
medium.
Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose appear
colorless on the medium and the agar surrounding the bacteria remains relatively
transparent.
Lactose can be replaced in the medium by other sugars and the abilities of gram-negative
bacteria to ferment these replacement sugars is [sic] detectable in the same way as is
lactose fermentation (for example Farmer and Davis, 1985).
Next week, we will provide you with a MacConkey agar plate containing Lac+ and Laccolonies for your inspection, so you can see the differences between these genotypes on the
media.
Mutagenesis methods
As stated above, geneticists are always on the lookout for interesting mutant phenotypes,
but given the low frequency of most of these mutations in nature, we often need to resort to
generating our own mutants in order to study a trait of interest. Your textbook outlines
mutagenesis and you should read those sections to get an overview of mutagenesis this
week. Next week, we will be using UV light to cause random mutations in the E. coli
genome. Cells naturally have the ability to repair damage to their DNA caused by UV light,
which reduces the efficiency of UV as a mutagen. As preparation for this lab, our colleague
Dr. Frank Healy has generated an E. coli strain in which he knocked out two of the genes
necessary for repair of DNA damage, (uvrA and phr), so that the cells are repair deficient.
We will use that uvrA- phr- strain to generate most of our mutant strains. We will also be
treating the parent strain with UV, and part of the results of this lab will be a comparison of
the DNA repair efficiencies of these two strains.
Prepare your lab notebook for the semester
You may record any lab notes in any lab-dedicated notebook. This can include notes on
discussions in lab, and ideas that you may have, but it MUST contain observations,
procedures and data – including sketches or photographs of bacterial colonies.
NOTE: if your lab notebook is a physical one, you must keep it in lab, or you can keep an
electronic lab notebook that is stored “in the cloud” (and thus not solely on one device).
If you are interested in keeping an electronic lab notebook, I suggest using the app
“GoodNotes” and will require that you upload your lab notebook to a secure file each week,
so that no work is lost.
Your notebook must have your identifying information in it, or saved to it. For example, if it
is a physical notebook, it must be labeled with your name, course & contact info. If it is
electronic, name it using the following format: First initial, Last name_BIO452L_SP17, e.g.,
RReynolds_BIO452_SP17
If using a physical notebook, each page must be labeled with a page number. Mistakes are
kept in the lab notebook and are simply crossed out. Do NOT tear pages out of your
notebook.
The first entry for each day should be preceded by the date
Each set of observations should have a title and a bit of explanation as to what your goal is
for the session, e.g., “Today we are observing UV-treated E. coli and sub-culturing colonies
that appear to lack the ability to metabolize lactose.”
Lab Work: Handling of bacterial cultures and dilution plating
Objectives and Overview
In this lab, we will be preparing for your investigative experiment by practicing pipetting,
aseptic, and bacterial culture techniques. These skills will be critical to your success
throughout the semester. Specifically, we will be learning how to perform dilution plating
of liquid cultures to get accurate counts of bacterial cell densities, how to streak from
colonies for isolates, and how to inoculate liquid media aseptically.
Lab Work Background 1: Dilutions and Dilution Plating
At times, the concentration of solute (or bacteria) in a solution may be too high for accurate
measurement. If this is the case, accurate dilutions of the solution can bring the
concentration into the necessary range, and the initial concentration can then be calculated
by measurement of the diluted sample multiplied by the dilution factor.
To dilute a sample, we take a known volume of the sample and add it to a known volume of
diluent (also known as a blank) that contains none of the solute of interest. The dilution of
the resulting sample is usually expressed as a fraction, and usually in whole amounts such
as 1/10 (spoken as “one to ten”), 1/100 etc., to make calculations easier. To calculate the
dilution, the amount of the sample is put in the numerator and the total volume (sample +
diluent) is in the denominator. For example, if we added 1 ml of sample to 9 ml of diluent,
the dilution is 1/10. The dilution factor (D) is the reciprocal of the dilution. In our example
above, the dilution factor is 10.
If we do not know the original concentration of a sample, it is not practical to do one
dilution and then hope that this concentration is within the acceptable range, nor is it
practical to do large-scale dilutions in one step (e.g., 1 M diluted to 1 μM). In these
conditions, we must dilute the concentrated solution several times in what is known as a
serial dilution to get the dilution(s) needed. Hopefully, one of these should result in a
measurable dilution, which can be used to calculate the amount in the original sample by
multiplying the measurement of the dilution times its dilution factor. A serial dilution is
illustrated on the following page.
There are some simple guidelines that you should follow when performing a serial dilution:
Label everything! It is easy to confuse which container is which.
Mix well! You will reduce your experimental error by mixing each solution or dilution well
before removing a portion for the next dilution. This is especially true with cells, because
they settle to the bottom of the tube.
Be careful with your pipetting. Make sure quantities are accurately and consistently
transferred from container to container.
Figure 1. Serial Dilution Scheme
Dilution
1/100
1/100
D=100
1/100
D=1x104
D=1x106
Each transfer results in a 1/100 dilution, so the sample in Bottle I has been diluted “100
fold.” Bottle II is a 1/100 dilution of Bottle I, and these dilutions are multiplied to get the
total dilution. If the original sample contained 8 x 108 cells/ml, Bottle I contains 8 x 106
cells/ml, Bottle II contains 8 x104 cells/ml, and Bottle III contains 8 x 102 cells/ml. Any
quantitative data obtained from a dilution, for example Bottle III, must be multiplied by the
dilution factor to determine the concentration in the original sample, and you can see that
calculating the original concentration using the reading from Bottle III gives the following:
8 x 102 x 1 x 106 = 8 x 108
Lab Work Background 2: Aseptic technique and manipulation of microbes
One of the keys to being successful in the lab when working with microorganisms is to
avoid contamination. This will be important for your experiments, so we will be practicing
transferring bacteria to plates and liquid broths.
General Procedures
During lab, you will make dilutions of a bacterial culture plate these dilutions onto agar
media. In addition, you will be streaking plates and inoculating broths.
Materials
•
Reagent 1
•
Reagent 2
Equipment
•
Equipment 1
•
Equipment 2
Procedure
Protocol 1: dilution plating of bacteria
For this protocol, you should work in groups of 4 to make one set of dilution plates.
Dilution: Make 10 mls of a 1:1000 dilution of the uvrA- phr- bacterial culture in Luria Broth
media and the antibiotic kanamycin (LB-Kan). In addition to being uvrA phr double
mutants, our bacteria are “Kanr” which means that they have an extra gene that has been
inserted that makes them resistant to kanamycin.
Dilution: Make 10 mls of a 1:100 dilution of your first dilution (what is the dilution factor
for this tube?)
Plating A: Plate 100 ul of the dilution made in (2) onto an LB-Kan plate labeled on the agar
half (not the lid) with your names and the letter 'A'. Note — it is easiest to observe what
happens on a plate if you label the plate using small but legible print around the edge of the
agar side.
Dilution & plating B: Make a 1:10 dilution of the dilution made in (2) and then plate 100 ul
of this new dilution onto an LB-Kan plate labeled with your names and 'B'.
Dilution & plating C: Make a 1:10 dilution of the dilution made in (4) and then plate 100 ul
of this new dilution onto an LB-Kan plate labeled with your names and 'C'.
Dilution & plating D: Make a 1:10 dilution of the dilution made in (5) and then plate 100 ul
of this new dilution onto an LB-Kan plate labeled with your names and 'D'.
Dilution & plating E: Make a 1:10 dilution of the dilution made in (6) and then plate 100 ul
of this new dilution onto an LB-Kan plate labeled with your names and 'E'.
Let the plates sit on the lab bench lid-side up until the bacteria are dry enough to store
upside-down. Then, incubate the plates upside-down in the 37C incubator in the
microbiology lab (WSC 118).
Each team member should come back after 12-24 hours and make a count of all the
colonies on a plate with a countable density. Record the data in your lab notebook with an
explanation of what they are. Use these data to calculate an estimate of the cells/ml in the
original tube, and include this in your notebook. Photograph the plated dilution series,
making sure that the colonies are countable and the plate label is legible in the photos.
Name the photos with your name and the date, and send them to your instructor by
uploading them.
Protocol 2: tube inoculation
Each person should complete this protocol independently.
Transfer 5mL of LB-Kan broth into a 15mL conical tube. Dip a flame-sterilized inoculating
loop into the Kanr liquid bacterial culture (uvrA- phr-), and then use it to inoculate the
fresh LB-Kan tube. Write your name and the word “inoculated” on the tube. Put the lid on
the tube, but make sure the lid is on loosely. Place the tube diagonally in the rack located in
the rotating incubator in the micro lab (WSC 118).
Transfer 5mL of LB (without Kan) into a second 15mL conical tube. Mock inoculate a tube
by going through the same procedure (flaming your loop, inserting it into broth, etc.),
except that in this case you do not dip the inoculation loop into the Kanr liquid bacterial
culture (this checks your aseptic technique). Write your name and the word “mock” on the
tube. Put the lid on the tube loosely. Place the tube diagonally in the rotating incubator.
Let them grow overnight, and check them the following day. Record in your lab notebook
whether the LB in each tube is clear (indicating no or low bacterial growth) or cloudy
(indicating bacterial growth). Take photos of the tubes, making sure the tube label is visible
in the photograph. Name the photos with your name and the date, and send them to your
instructor by uploading them to this address:
https://www.dropbox.com/request/9M5w8RockdlJFnvaZqui
If you are keeping a physical lab notebook, your instructor will provide you with printed
photos to insert into your physical notebook as soon as possible. Be sure to leave space in
your notebook for those photos. If you are keeping an electronic notebook, insert the
photos you took, using tape or acid-free glue. Whether you are keeping a physical or an
electronic lab notebook, you must give each photo a legend that explains what it is.
Please do not dispose of these liquid cultures. Photographs aren’t always clear enough to
show growth or lack of growth, so your instructor will be examining your tubes in person.
Protocol 3: streaking for isolates
Each person should complete this protocol independently.
The following protocol has been adapted from http://www.sciencebuddies.org/sciencefair-projects/project_ideas/microbio_streakplates.pdf .
Protocol 3 Overview
In order to obtain well-isolated, discrete colonies, the quadrant-streak technique can be
used. This allows sequential dilution of the original microbial material (broth culture or
colonies on a plate) over the entire surface of a fresh plate. As streaking the microbes over
successive quadrants of the plates dilutes the original sample, the number of individual
organisms decreases. Usually by the third or fourth quadrant only a few organisms are
transferred on the inoculating loop and these produce a few isolated colonies. Study the
diagram and the steps below before you begin.
Flame the inoculating loop until it is red hot and then allow it to cool.
Use the sterile inoculating loop to remove a single colony from the source bacteria plate OR
if using liquid culture, dip the inoculating loop into the broth culture.
Immediately streak the inoculating loop very gently over a quarter of the plate using a back
and forth motion (see area 1 in Figure 2 below).
Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just
streaked, extend the streaks into the second quarter of the plate (area 2, Figure 2).
Flame the loop again and allow it to cool. Going back to the area that you just streaked
(area 2), extend the streaks into the third quarter of the plate (area 3).
Flame the loop again and allow it to cool. Going back to the area that you just streaked
(area 3), extend the streaks into the center fourth of the plate (area 4).
Protocol 3 Specific instructions: Streak Kanr and Kans bacteria
Using aseptic technique, get a loopful of bacteria from the culture of Kanr bacteria provided
and streak out for isolates on a LB-Kan plate. Label the plate with your name and a code
that will allow you to identify your plate and place it in the 37C incubator.
Next do the same procedure with the Kanr culture and an LB-Amp plate (these are agar
plates made with Luria Broth and the antibiotic ampicillin).
Next do the same procedure with the Kanr culture and a plate of LB agar with no added
antibiotics.
Finally, do the same procedures with the Kans culture (this is the kanamycin susceptible,
wild type bacteria) and an LB-Kan, LB-Amp, and an LB plate.
Incubate these plates overnight in the 37C incubator.
Come back after 12-24 hours and make a count of all the colonies on a plate with a
countable density. Record the data in your lab notebook with an explanation of what they
are. Photograph the streaked plates, making sure the cultures and plate labels are visible.
Name the photos with your name and the date, and send them to your instructor by
uploading them.
If you are keeping an electronic notebook, insert the photos you took, using tape or acidfree glue. Whether you are keeping a physical or an electronic lab notebook, you must give
each photo a legend that explains what it is.
After counting the colonies on your plates, be sure to dispose of the plates in the biohazard
box. (Your instructor will be looking at your plate series photographs to grade for
technique).
Recipes
List recipes for solutions and medium.
References
Bibliography
Provenzano D., Schuhmacher D.A., Barker JL, Klose KE. 2000. The virulence regulatory
protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic
Vibrio species. Infection and Immunity. 68(3):1491-7
Thanassi, D. G., Cheng L. W., and Nikaido, H. 1997. Active efflux of bile salts by Escherichia
coli. Journal of Bacteriology. 179:2512-2518
Farmer, J. J. 3rd, and Davis, B. R. 1985. H7 antiserum-sorbitol fermentation medium: a
single tube screening medium for detecting Escherichia coli O157:H7 associated with
hemorrhagic colitis. Journal of Clinical Microbiology. 4:620-5.
Healy, F. G., Livingstone, K. D. 2010. Using student-generated UV-induced Escherichia coli
mutants in a directed inquiry undergraduate genetics laboratory. Genetics. 186:33-39.