Professional Documents
Culture Documents
Jimenez 2
TABLE OF CONTENTS
I. Acknowledgements .1
II. Purpose ..2
III. Hypothesis.3
IV. Review of Literature 4-11
V. Materials & Equipment ...12-13
VI.Procedures & Protocols ....14-22
VII.Variables ..23
VIII.Data & Results ..24-31
IX.Data Analysis .32
X. Experimental Error .33
XI.Conclusions & Societal Impact ..34-36
XII.Reference List .37-38
Jimenez 1
Acknowledgements
This paper is dedicated to my family, for their continuous support and their help. I hope
they will always be encouraging in all the events and milestones ahead in my life. I would like to
thank The University of Chicago for being very hospitable during the couple weeks I was there
for my research. To Doctor Beatrice Fineschi, I thank her for all of her suggestion and for her
help in improving my research experiment. I would also like to thank her for introducing me to
the wonderful world of Microbiology and Immunology, and for this I am truly grateful. To Dawn
Sweeney, I thank her for being a spectacular Teachers Assistant. Thanks for everything and for
the life lessons you helped teach me. To The University of Chicagos Sequencing and
Genotyping facility, thank you for mapping out my protein. I also thank Ms. Camel for being an
amazing SIRS teacher, for helping me along the way, and for printing out all of the materials I
need for my research project. I would also like to thank her for editing my Review of Literature,
giving detailed analysis and sending me reminder updates for the project.
Jimenez 2
Purpose
Antibiotic Resistance is created towards rifampicin resistance with a 4-methyl-1piperazinaminyl group using DH5 Escherichia coli strain. This is used to determine how the
rifampicin inhibited the bacteria from performing binary fission and eventually causing it to
undergo cell apoptosis. Transcription factors are also looked with the bacterial RNA Polymerase
and analyzed using a Polymerase Chain Reaction and a sequencing and genotyping facility at
The University of Chicago. The rpo gene will be used to determine the allosteric sites of
inhibition causing the mutations within the beta subunit pocket groove. After sequencing the
gene, the mutated DNA from the rifampicin resistant bacteria will be compared to the wild type
DH5 Escherichia coli strain and analyzed for points of mutations using Sequencher.
Jimenez 3
Hypothesis
Hypothesis 1
If DH5 E. coli is exposed to rifampicin, an antibiotic known to impede on transcription and
RNA Polymerase, then the DH5 E. coli will survive because DH5 E. coli has the ability to
mutate rapidly around the points of mutation, and as the concentration of rifampicin increase, the
colonies should decrease.
Rationale: Mutations within the rpo will cause the pocket (groove) of the bacteria to warp as it
will code for different proteins. This change of protein is enough to directly alter the
intermolecular forces that exist within the structure and the shape of the structure. With the
change of shape, rifampicin cannot bind as well in the receptor site causing the E. coli to
continue replicating.
Hypothesis 2
If the DH5 E. coli survived because of the rapid mutations, then after DNA sequencing and
protein modeling, it will be found that the allosteric site of inhibition will be changed due to one
point mutation to cause the pocket groove.
Rationale: The changes of protein cannot happen simultaneously in an allosteric groove site. The
proportion of change over the three day span is not enough to cause multiple mutations within
the rpo subunit of the protein. Also it is highly unlikely to have more than one point of mutation
because the rpo is a well preserved DNA fragment of the E. coli showing that specific changes
in this gene will allow it to undergo a microevolution.
Jimenez 4
Review of Literature
Through the process of evolution, many species arose from a common ancestor and made
Earth their homes. Over time as these species received genetic mutations in their genomes, they
created different organisms. Charles Darwin's once said, One general law, leading to the
advancement of all organic beings, namely, multiply, vary, let the strongest live and the weakest
die. (Darwin, 1996). In his Theory of Evolution he is implicating in essence that some cells that
are not suitable to adapt will die. In this experiment, DH5 E. coli is exposed to Rifampicin a
known antibiotic that inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial
DNA-dependent RNA polymerase (Bethesda, 2011). The experiments' goal is too see whether or
not E. coli can mutate and adapt around Rifampicin and by doing so it has to change its proteins
shape where the Rifampicin binds to it in the rpo Beta subunit.
Mutations make it possible for organisms to
adapt and survive the obstacles present to them and to
continue making progenies. In this sense E. coli's rate
of mutations actually occur faster that human DNA.
There is usually one mutation per 10^7 amount of
bases copied in DH5 E. coli as compared to human
DNA which is 10^8 (Drake, 1998). This makes sense
as bacterial genomes are far greatly smaller in number
than human DNA as well as the fact that eukaryotes are more complex than E. coli. In
fact ,bacteria can replicate every twenty minutes showing that the process of binary fission
occurs much more rapidly than the rate of mitosis in humans. Again, this is because their genes
Jimenez 5
are smaller and contain everything vital for the bacteria, the rest are located in its plasmids where
nonessential genetic information are stored (Fineschi, 2014).
Rifampin specifically inhibits bacterial RNA polymerase, the enzyme responsible for
DNA transcription as mentioned previously. So the only way for E. coli to survive is if exposed
to Rifampicin and if it changes its protein structure within the beta sub unit of its RNA
Polymerase. However, it does not do so because of Rifampicin, and because the E. coli mutated
as mutations occur randomly. By changing its structure, Rifampicin cannot properly bind along
the receptor sites making the E. coli strain resistant to it and continue replication and making
proteins. This is because RNA Polymerase plays a crucial role for the bacteria. It uses the DNA
found in E. coli as a template and transcribes them in the promoter region of the cell. Through
the Central Dogma of Biology, DNA is converted into RNA through the process of transcription
and then it is converted into proteins through translation (Gerstein, 2007). So this shows that if
Rifampicin inhibits RNA Polymerase,
transcription cannot take place and so the
bacteria cannot make more proteins and will
Photo 2.0 shows the basic structure of the
Central Dogma Theory of Molecular Biology
lyse.
Jimenez 6
enables transcription initiation, and this is followed by elongation of the transcript. In turn,
transcript elongation leads to clearing of the promoter, and the transcription process can begin
yet again. Transcription can thus be regulated at two levels: the promoter level (cis regulation)
and the polymerase level (trans regulation). These elements differ among bacteria and eukaryotes
however. In this experiment, the rifampicin binds to the rpo gene that encodes the subunit of
bacterial RNA polymerase.
The binding into the rpo Beta sub unit of E. coli will cause a mutation in this area. It
will also cause the protein structure to bend or arrange itself differently making it hard for the
Rifampicin to bind as easily as it could without the mutation (Martinez, 2001). One mutation is
enough to alter the shape of the bacteria. In fact there are two types of interactions that could
occur: direct or indirect interaction. Direct interaction is if the rotamers around the protein region
bind directly to the site causing movement between the two molecules. Indirect interaction is if
the rotamers around the protein region do not necessarily bind together but however has an
influence around the region due to its electronegative state. Also the idea of Steric Hinderance
arises because if atoms are rearranged it might affect the preferred shape of the molecule and
have different amounts
of rotamers based on the
neighboring molecules
(intermolecular forces)
Photo 3.0 shows the different types of interactions in a simple ELISA
diagram, the various interactions affect the molecule in distinct ways
protein (Reusch, 2013) This is important to note because one shift can cause a chain reaction of
Jimenez 7
angle bends along the molecular level warping the structure from the original shape. This bent
phenomenon can be seen by observing the rpo Beta sub unit of E. coli.
Specifically, the rpo Beta sub unit of bacteria is what is classified as a preserved gene
sequence. This means that as the bacteria grows over time, many parts and features can change
that give rise to new types of bacterias and strains but the idea of sequences being preserved is
that it is very important for the bacteria to survive. Again this is because the rpo Beta sub unit
contains instructions for RNA Polymerase to divide and replenish DNA. With an inhibition of
this part it can no longer divide. Alterations in this gene might also lead to cell apoptosis
considering that this is important in survival. The mutated genes that will be found that causes
rifampicin resistance may lead scientists to see what sequences within the preserved sequence
can the bacteria live without. This however might cause complications for the bacteria such as
slower movement and growth overall, leading the wild type strain to be better off in competition
in confluence levels if there are no rifampicin or antibiotics present that is derived from
Rifamycin (Fineschi, 2014).
The wild type strain is something that prevails amongst the different types of the same
species. In general terms it is distinct from other
mutant types, it can be seen as the normal and
average strain. It is only through the exchange of
DNA that allows it to derive from the wild type. There
are two specific ways bacteria can pass on its genes
and lead to alterations and mutations, the Vertical Gene
Transfer which is the transfer of DNA from the parent
Jimenez 8
to the daughter cells when the bacteria undergoes binary fission, and through Horizontal Gene
Transfer. Specifically Horizontal Gene Transfer plays a crucial role in DNA exchanges when
talking about antibiotic resistance. There are three main types of this transfer: transformation,
transduction, and bacterial conjugation. Transformation occurs when the bacterial receptor sites
phagocytose floating pieces of DNA and attach it to its own plasmid loop. Transduction occurs
when one bacterium or a phage transfers its
DNA specifically to another bacterium.
Bacterial conjugation happens when DNA
gets transferred through a cell to cell contact
usually recombining the DNA. (Fineschi,
2014). These phenomenon of mutations from
the wild type can easily be observed in the
water.
Lake Michigans fauna makes it one
of the more diverse ecosystems in the world. Containing everything from birds, fish, moose all
the way to algae and bacteria (Prescott, 2003). Not only that but extensive changes in weather,
climate, and temperature in Chicago makes it easily accommodating to many species. In fact on
a sunny day in still water, bacteria can actually germinate rapidly making people wonder whether
or not the water is really safe to drink. However in the 21st century, water purification is of a
high priority. The City of Chicagos Water Treatment plant filters the lake water through eight
traveling screens, where it is then chemically treated in a flocculation process, where it is then
allowed to settle. The next day this water is then refiltered, poured in mixing basins for another
Jimenez 9
chemical treatment before finally making its way to pipes and various showers and faucets
around Chicago (Wheat, 2014). With this much said, the water in Chicago is already more clean
compared to other water sources in the world, yet the city makes a lot of effort to ensure that the
water is drinkable to that extent. This is because bacteria can still be lurking and mutating
(Hodgson2012). In fact, Coliforms and E. coli make their homes in the waters so regardless how
clear the water looks, one would simply not drink the water straight from the lake. Birds and
other marine animals suffice to say when they expel their fecal waste, they also expel the bacteria
living in the gastrointestinal tracts. EPA states that for Lake Michigan in the part of Illinois to be
considered safe, it must not exceed 100 Fecal Coliforms/ml (EPA, 2003). In fact EPA also states
that 99% of the water is monitored by the E. coli living there. It is constantly sequenced and
monitored for changes in sequencing to check if pathogens may be lurking in the waters.
Scientists use the 16S ribosomal RNA found in E.
coli as it is known to be a highly conserved part in
the bacteria. The 16S rRNA helps bind together the
two ribosomal subunits of 50S and 30S by
interacting with 23S. In essence, scientists who use
E. coli to monitor the water also developed a method
through PCR that amplifies the bacterial genomic
sequences. In doing so they have developed many
Photo 6.0 shows the 16S rRNA, and the two
ribosomal subunits of 50S and 30S
different segments of the 16S gene. One reason why scientists like to use this gene is because
they use it to form ancestral phylogenetic tree of different species of bacteria and the evolved
Jimenez 10
archaea. In this watery environment, bacteria can thrive and easily exchange DNA from one
bacterium to another. By looking at sequences, they can tell right away what type of strain and
type of bacteria lurks in the waters by performing a quick genetic coding test (Markson, 2013).
Genetic sequencing helps to identify the organism right down to its nucleotide sequence
structure. Genetic sequencing also helps develop new and powerful medicine that can be
personalized according to the genetic makeup of a person as can be seen in the Human Genome
Project. In fact, with a Polymerase Chain Reaction (PCR), DNA segments can easily be
sequenced
to thousands of
The follow
up of gel electrophoresis to
sort out DNA by sizes and
ExoSAP procedures help
purify the specific locus of
DNA that is being currently investigated (Feingold, 2011). The rise of genetic sequencing
specifically helped scientists find mutation points, and this technique is used not to classify the
bacteria in this experiment but to replicate the rpo Beta subunit of a wild type and a rifampicin
resistant mutated E. coli. After the genetic sequences are coded, it can be easily told what part in
the genes caused the antibiotic resistance to happen. This is because three nucleotides make up a
codon which will code a specific amino acid gathered by the tRNA. Essentially a mutation
within the nucleotides will almost always result in a mutation in the protein structure as amino
acids are the building blocks of protein. With this interaction, the one protein misshape may
Jimenez 11
cause the primary, secondary, tertiary, or quaternary structure to reconfirm making it hard for
specific epitopes like the one in rifampicin to easily bind into the rpo Beta subunit.
Looking at the structure of the protein more closely, changes of specific enzymes can be
easily seen by modeling at the molecular level. Nowadays, it is even easier to tell where the
obstruction takes place by pasting the sequence in a protein modeler analysis. Protein modeling
allow scientists to study interactive forces, and charges that case some proteins to behave the
way the do (Fineschi, 2014). Overall the big
picture can be seen, and after modeling the
structure being looked at compared to the structure
that does the interaction, it can be clearly depicted
if binding is occurring or not. This program was
used in this study to look at the orientation of the
rifampicin against the rpo Beta subunit of the E.
coli.
Jimenez 12
40 petri dish
5g NaCl
5g Tryptone
2.5g yeast extract
7.5g Agar
Deionized H2O
Rifampicin
Pipettes
Stir Rod
Erlenmeyer flask (500 ml)
Hot Plate
PBS
Test Tube Racks
Test Tubes
Light Microscope
Selecting and lysing a mutant colony
Jimenez 13
Thermocycling and PCR of the mutant
Sequencing
1 mL Taq polymerase
A computer
Thermocycler
Excel Sheet
Centrifuge
A camera
Gel electrophoresis
1% Agarose Gel
Microvials 10 l
PCR Samples
10% ExoSap
40 Pipette Tops 10 l
10 Pipetteman 10 l
PBS
Electrodes
Voltage Source
Casting Trays
(Ethidium Bromide)EtBr 0.5g/m
Microwave
Jimenez 14
Jimenez 15
Procedure:
1. The diagram depicts the serial dilutions you need to do.