Aria Foroughi

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Nature of Mutations Homework Questions

1) The Lenski experiment was carried out by Richard Lenski and his crew, who used controlled
lab conditions to observe and research the evolutionary process. He carried out long-term
evolution research through his experiments and demonstrated how organisms change and
adapt over time(Crozat et al., 2005).

Lenski started his experiment in 1988 with a colony of 12 E. coli bacteria, which were cultured
by being separated into flasks that contained nutritional agar media. They move a little fraction
of the cells from the flasks to the new medium to generate the continuous growth of cells. For
over three decades, it has been the longest experiment conducted. The study of evolution
across time is aided by this. The researchers closely monitor a variety of experiment-related
factors over this time, including growth rates, genetic alterations, and laboratory environment
adaptations. Indeed, they carried out the tests by allowing cells to develop in various
environments. This helps them comprehend the fitness analyses by which bacterial strains
demonstrate reproductive success in diverse environments and, as a result, aids in
understanding the selection benefit of various mutations taking place. Gene sequencing was
employed to analyze the genetic alterations that occurred over time, and samples of cells or
strains were occasionally kept in the freezer for future research(Crozat et al., 2005).

• This is how the LTEE experiment works: A daily transfer of 1% of the inoculum from each of
the 12 populations was made into a new DM25 growth medium while the populations were
incubated at 37°C.
• Data from the LTEE experiment, which was conducted and is currently ongoing, has been
used to track changes in bacterial populations. The astonishing outcomes of this experiment
include changes in fitness, cell size, evolution, dietary demand, and population variance.
• This extensive and well managed experiment has opened a lot of doors for many scientists
and in their research generally over the past few years. This experiment has been ongoing since
1988, thus there must be some results that are important for comprehending microbiological
growth and changes that take place with time(González Casanova et al., 2015).

In other words, The E. coli long-term evolution experiment (LTEE), directed by Richard Lenski,
has been studying genetic changes in 12 populations of asexual Escherichia coli bacteria that
were initially identical. The long-term evolution experiment was created as an unrestricted way
to examine key aspects of evolution experimentally. The experiment set out to investigate the
dynamics of evolution, notably the pace of evolutionary change, as well as the repeatability of
evolution and the link between phenotypic and genotypic changes (Richard Lenski - evolution in a
flask 2017).
 a. The experiment was conducted to check the long-term adaptations and evolutions of population of 12
E. coli, by means genetic analysis, and fitness analysis(Callaway, 2022).

2) Bacteria generate genetically identical offspring through asexual reproduction. Bacteria must
undergo a genetic shift that results in a better suited phenotype to survive the hostile
environment. Mutations enable those genetic alterations.
An experiment that simulates the development of bacteria toward antibiotic resistance has
been designed by researchers from Technion-Israel Institute of Technology and Harvard
Medical School. Microbial Evolution and Growth Arena (MEGA)-plate is the name of the
experiment(MEGA-Plate Experiment - Labster Theory, n.d.).

Furthermore, On the portions of the plate without any antibiotics, the bacteria are first
developed. The bacterium colony eventually covers all the regions without antibiotics after
several hours. Before the bacterium colony can transfer to the following plate portion with a
10X antibiotic concentration, growth then slows down. Until the bacteria colony can get to the
plate region with a 1000X antibiotic concentration, the process is repeated. The entire trial
lasted around two weeks.
The trials demonstrate that bacteria may quickly change and develop resistance to high-
concentration antibiotics(Khan Academy, 2021).
Moreover, the emergence of drug resistance, a significant issue happening in the practice of
medicine, is shown by the Kishnoy mega-plate experiment. A Markov Process or Markov Chain
is the name for the evolutionary process being carried out by the microorganisms in this
experiment. Clinicians and researchers can forecast the development of drug resistance and
create plans to stop it by comprehending this process. In other words, when it comes to
evolution, bacteria have numerous benefits over their human hosts. To start, their generation
periods are considerably shorter, which speeds up the selection of useful alleles within a
population(Kleinman, 2021).
Images 1-5: Based on Harvard Medical School's The Evolution of Bacteria on a "Mega-Plate" Petri Dish. In just 12
days, E. coli developed the necessary resistance to withstand 1,000 times as many antibiotic as it could have at the
beginning of the trial(Strange Science_ What Is Evolution_, n.d.).

3)

A) The current experiment shows the bacterial plates grown on the agar plate with different
volumes of dilutions. As dilution is increased the count of bacteria growing on the agar plate
decreases.

• The experimental E. coli strain can consume several plasmids with different antibiotic
labels. When the E. coli bacteria are cultivated on agar plates with ampicillin added to
the agar, the plasmids confer antibiotic resistance in the bacteria. The ampicillin
degrading enzyme, which breaks the beta-lactam ring of the ampicillin antibiotic, is
produced, and secreted by the E. coli bacteria after the plasmid was inserted into them
during the transformation process. As a result, the ampicillin beta-lactam ring is broken
down, allowing the bacteria to thrive on the ampicillin-containing E coli plate.

• Ampicillin's beta-lactam ring, which is present in this antibiotic, suppresses bacterial

growth when it is applied to an agar plate, which is how it works. The mechanism of
action of ampicillin involves inhibiting the synthesis of the peptidoglycan layer in the
bacteria. The cells become resistant to ampicillin because the gene present in the
plasmid inside the E. coli strain secretes the beta-lactam ring destroying enzyme,
causing these bacteria to become resistant on the agar plate (Ampicillin - PubMed, n.d.).
A:
Aria Foroughi/E. coli DH5α /Negative Control (-)
B:
Aria Foroughi/E. coli DH5α /Negative Control ampicillin 5mg\ml
C:
Aria Foroughi/E. coli DH5α /Negative Control ampicillin 10mg\ml
D:
Aria Foroughi/E. coli DH5α /Negative Control ampicillin 20mg\ml
E:
Aria Foroughi/E. coli DH5α /Negative Control ampicillin 100mg\ml

B) The plate with 0 ug/ml, or no ampicillin, will have a greater number of colonies.
Because bacterial growth will not be suppressed in the absence of ampicillin (in the
plate), there will be more colonies on the plate, as can be seen in the plate marked
(a). The number of colonies on the plate in this instance is incalculable. Then, when
the ampicillin concentration rises, the number of colonies will decrease. Less
bacterial colonies are seen on the plate as the concentration of ampicillin rises
because the development of bacteria is hampered(Ampicillin - StatPearls - NCBI
Bookshelf, n.d.). Additionally, the plate with the label (e) has no colonies at all since
it contains the most ampicillin, i.e., 100 ug/ml. So, the correct order of the plate with
0 to 100 ug/ml concentration of ampicillin is a, b, c, d, and e.
  In addition to our lab photos which analyzed above, I also found typical images of the
colonies grown on the agar plate inoculated with the methicillin-resistant
Staphylococcus aureus (MRSA) suspension after treated by the bare K-wires (a and b)
and the PL-coated K-wires (c and d) for 1 h (a and c) and 24 h (b and d)(Typical Images
of the Colonies Grown on the Agar Plate Inoculated With, n.d.).

C)The synthesis of B-lactamase enzymes by bacteria is the main mechanism for ampicillin
resistance. Ampicillin is a member of the group of antibiotics known as -lactam antibiotics,
which work by preventing the formation of bacterial cell walls in their target
organisms(Ampicillin - PubMed, n.d.). Ampicillin and other B-lactam antibiotics are susceptible
to having their B-lactam ring structure destroyed by B-lactamase enzymes, leaving them
useless.

Furthermore, Bacteria can develop -lactamase enzymes when they acquire the genes for these
enzymes through horizontal gene transfer or mutation. The B-lactamase enzymes are in the
periplasm or released into the bacterial cell's environment where they can interact with
ampicillin molecules. The ampicillin's -lactam ring is subsequently hydrolyzed by enzymes,
rendering it inactive and making bacteria resistant to its bactericidal actions(Mora-Ochomogo &
Lohans, 2021).
It's significant to remember that while the formation of B-lactamases is the most typical
mechanism of ampicillin resistance, other processes may also be involved in some bacteria.
These methods include ampicillin efflux pumps, which actively pump the antibiotic out of the
bacterial cell, changes to penicillin-binding proteins (PBPs), which lessen ampicillin's affinity for
binding to them, and changes to the permeability of the bacterial cell wall, which restrict
ampicillin entrance. However, the predominant and most common pathway for ampicillin
resistance continues to be the development of -lactamases(Kapoor et al., 2017).

References:

Ampicillin - PubMed. (n.d.).

Ampicillin - StatPearls - NCBI Bookshelf. (n.d.).
Callaway, E. (2022). Legendary bacterial evolution experiment enters new era. In
Nature (Vol. 606, Issue 7915, pp. 634–635). https://doi.org/10.1038/d41586-
022-01620-3
Crozat, E., Philippe, N., Lenski, R. E., Geiselmann, J., & Schneider, D. (2005). Long-
term experimental evolution in Escherichia coli. XII. DNA topology as a key
target of selection. Genetics, 169(2), 523–532.
https://doi.org/10.1534/genetics.104.035717
González Casanova, A., Kurt, N., Wakolbinger, A., & Yuan, L. (2015). An individual-
based model for the Lenski experiment, and the deceleration of the relative
fitness. Stochastic Processes and Their Applications, 126(8), 2211–2252.
https://doi.org/10.1016/j.spa.2016.01.009
Kapoor, G., Saigal, S., & Elongavan, A. (2017). Action and resistance mechanisms
of antibiotics: A guide for clinicians. In Journal of Anaesthesiology Clinical
Pharmacology (Vol. 33, Issue 3, pp. 300–305).
https://doi.org/10.4103/joacp.JOACP_349_15
Khan Academy. (2021). Bacterial transformation & selection (article) | Khan
Academy.
Kleinman, A. (2021). The Kishony Mega-Plate Experiment, a Markov Process. In
bioRxiv (p. 2021.12.23.474071).
https://www.biorxiv.org/content/10.1101/2021.12.23.474071v1%0Ahttps://
www.biorxiv.org/content/10.1101/2021.12.23.474071v1.abstract
MEGA-plate experiment - Labster Theory. (n.d.).
https://theory.labster.com/mega_plate/
Mora-Ochomogo, M., & Lohans, C. T. (2021). β-Lactam antibiotic targets and
resistance mechanisms: From covalent inhibitors to substrates. In RSC
 Medicinal Chemistry (Vol. 12, Issue 10, pp. 1623–1639).
https://doi.org/10.1039/d1md00200g
Strange Science_ What is Evolution_. (n.d.).
Typical images of the colonies grown on the agar plate inoculated with. (n.d.).

Richard Lenski - evolution in a flask. YouTube. (2017, February 24).

https://youtu.be/PQr8ldEeO04