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pGLO™ Transformation and Inquiry Kit

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Science Case Study

Contents

Science Case Study — “Hacking the Gut Microbiome: Could Engineered Bacteria Relieve Dysentery
Symptoms?”........................................................................................................................................................... 1

Bibliography........................................................................................................................................................... 8
Science Case Study
“Hacking the Gut Microbiome: Could Engineered Bacteria Relieve Dysentery Symptoms?”
Part 1 — Setting the Scene.
Dr. Karen Cruz felt like she hadn’t slept for a week. Running back and forth from one bed to another in the hot and arid climate
of Afghanistan had taken its toll on her. In a far-flung military outpost outside Kandahar, she had been tending to the soldiers for
several weeks after a dysentery outbreak gripped the troops. Even her nursing staff had succumbed to the illness, and she was
short staffed to begin with. Now, sitting at her desk, she began to review her notes:

January 21

Today 15 more soldiers have presented symptoms of what I suspect

UZBEKISTAN
is a quickly spreading bacterial infection that causes dysentery.
T U R K M E N I S TA N
Symptoms include fever, chills, bloody diarrhea, loss of appetite,
TA J I K I S TA N

and dehydration. The total count on bed rest was 56, plus these 15
Sheberghan
make 71, with only 30 beds in our medical tents. Keeping everyone Herat
Konduz

quarantined from the remaining 75 personnel on base is more A F G H A N I S TA N

IRAN Kabul
and more difficult. Farah
Jalalabad

Zaranj
Kandahar
Luckily there have been no casualties, since we are well stocked
with IV fluids to keep the most gravely ill hydrated. However,
PA K I S TA N

I wonder if there is anything that can be done to relieve their

symptoms. I can tell the soldiers are doing their best to keep a
positive attitude, but morale is beginning to diminish.

Because it was nearly impossible for the troops at the outpost to avoid infection from the local food and water, Dr. Cruz hoped
maybe she could at least diminish the symptoms and help her patients recover more quickly. Dr. Cruz kept up to date with medical
research and noted that there has been a lot of recent interest in microbiomes — the collective genomes of all the microorganisms
living in and on an organism, including a human, and how they influence the outcome of acute and chronic illnesses. She also
remembered that one of her medical school professors was involved in research funded by Defense Advanced Research Projects
Agency (DARPA) to synthesize genetically engineered bacteria to combat gastrointestinal diseases like dysentery. She decided
to email her former professor to share her experience and find out more about his research. She was pleasantly surprised by his
response:

Dear Dr. Cruz,

Thanks for reaching out to me and thank you for supporting our troops! The initial phases of
my research have proven quite successful and may be of use to you. Our research question is:
Can genetically engineered bacteria be used to reduce the symptoms of dysentery? We decided
to genetically engineer E. coli using transformation as a method to program the E. coli to
sense infection in the gastrointestinal tracts of mice and then stimulate the mouse’s own
immune system to fight the infection faster than it normally would—so the mice would be sick
for half the normal time. Our hypothesis is that it is possible to 1) engineer bacteria to
detect infection and 2) engineer the bacteria to induce a host immune response faster than
it would naturally occur once the engineered bacteria detected infection.

I’ve attached our initial data here with further explanation. Let me know what you think.

Sincerely,

Dr. Richardson

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Dr. Cruz was excited to hear of the advancements in Dr. Nurse Brady: Yes, exactly. It’s true that some bacteria can
Richardson’s research. She hoped that this work would allow cause illnesses such as dysentery when they are present in
troops in the future to recover from dysentery sooner or even high numbers in the gut. They can do this in several ways.
prevent them from becoming ill. Dr. Richardson’s email made Some produce toxins that cause inflammation of the intestinal
sense, but there was still a lot Dr. Cruz did not understand lining, resulting in diarrhea. Others may cause ulcers to form
about genetically engineered bacteria: how they are made in the intestines, resulting in blood in solid waste. Some E. coli
and how they work in the mice’s intestines. Before looking strains are harmful, but other strains are not, particularly those
at the data, she decided to do a little research of her own to used in research labs like E. coli HB101, which are engineered
determine whether the engineered bacteria would be useful to so that they are not harmful. Dr. Richardson must be making
treat dysentery. She would need to understand the process of use of these harmless E. coli strains.
engineering the bacteria. She decided to consult with her head
nurse, Ms. Brady, who had a background in microbiology and Dr. Cruz: That makes sense. Can you tell me more about
genetics. microbiomes? I have read a little, but I gather that you have
been reading much more on the topic.

Nurse Brady: Well, the first thing to consider is that

mammals, including humans, have more bacterial cells in their
gastrointestinal, or GI, tracts than the number of cells making
up their entire bodies. There are up to 100 trillion bacteria in
your GI tract and approximately 37 trillion cells that make up
your body. As you know, the GI tract includes the series of
organs through which food passes, nutrients are absorbed,
and solid waste is eliminated. Bacteria live on the surfaces of
the GI tract and these communities, made of many different
types of bacteria, are referred to as your GI tract’s microbiota.

Dr. Cruz: Nurse Brady, I’m really optimistic that Dr.

Richardson’s research can help in the fight against bacterially
transmitted diseases like dysentery. But there are still several
things I don’t fully understand about his team’s research. I’m
hoping you can help me. First off, could you give me a refresher
on the process of bacterial transformation? Several biotechnology companies are investigating how those
bacteria can influence our overall health, how where we live can
Nurse Brady: Sure, I’m glad to help! Genetic transformation determine what types of bacteria make up our microbiota, and
in bacteria is a technique that scientists use to insert genes into how what we eat (or don’t eat) can influence the composition
an organism, in this case the bacterium E. coli. In addition to of our microbiome. When bad bacteria or viruses grow in
one large chromosome, bacteria naturally contain one or more number in your GI tract and you get sick, you can think of the
small circular pieces of DNA called plasmids. These plasmids gut microbiota as being out of balance. The purpose of adding
can be re-created by scientists to include genes they want to these genetically engineered E. coli is to allow the immune
express in the bacterial cells. In the case of Dr. Richardson’s system to more quickly respond to the infection and rid the
research, it sounds like they plan to transform E. coli with a body of the harmful bacteria. This would then allow the normal
plasmid expressing a gene that triggers the mouse’s immune balance of microbiota in the gut to reestablish itself.
system when infection is present.
Dr. Cruz: Thanks so much. That helps clear things up for
Dr. Cruz: I see. So Dr. Richardson and his team plan to me. Let’s take a look at the data Dr. Richardson shared and
transform the E. coli to express a gene that it normally wouldn’t, determine whether we think his work could help to reduce the
and that may help mice recover from dysentery more quickly. number of days that patients experience dysentery symptoms.
Another thing that Dr. Richardson mentioned is that the Maybe this research will eventually be useful in reducing the
engineered E. coli can help combat the “bad” bacteria and severity as well as the duration of dysentery symptoms in
viruses in the gut. humans. I see his email attachment starts with a note.

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Part 2 — In Vitro Experimental Results.

Dear Dr. Cruz,

Please see the latest data we gathered during trials with mice that have dysentery. We
conducted two experiments to determine 1) how the presence or absence of inducer molecules
similar to those associated with gastrointestinal infections would affect expression of the
reporter gene firefly luciferase and 2) if engineered E. coli expressing an immune-stimulating
gene could reduce the number of days mice exhibit symptoms of dysentery.

We began by designing promoters that would activate expression of a reporter gene in

response to gut inflammatory signals. GI infection produces inflammatory signals that induce
these promoters. The reporter gene we used is the luciferase gene, which encodes an enzyme
found in fireflies that generates light when it cleaves its natural substrate, luciferin.
We cloned multiple versions of the promoter into a plasmid that also had an antibiotic
resistance gene, and an origin of replication. We decided to test two different versions of
the promoter to see which version would be most effective, A or B (Figure 1).

Gene of interest Gene of interest

(firefly luciferase reporter gene in (firefly luciferase reporter gene in
experiment 1 and immune-stimulating experiment 1 and immune-stimulating
gene in experiment 2) gene in experiment 2)

Promoter A Promoter B

Ori Ori

Antibiotic resistance gene Antibiotic resistance gene

Fig 1. Schematic of plasmids used in transformation studies. For experiment 1, the gene of
interest is firefly luciferase gene. For experiment 2, the gene of interest is an
immune-stimulating gene.

Experiment 1:

The purpose of this experiment was to test whether the newly designed promoters are induced
by inflammatory signaling molecules and to compare the level of induced gene expression. In
order to do this we transformed gut-derived E. coli from mice with the plasmid containing
Promoter A and the firefly luciferase reporter gene (Strain A) or the plasmid containing
Promoter B and the firefly luciferase reporter gene (Strain B). We measured the amount
luciferase expressed downstream of the promoter by performing an assay to measure the amount
of light produced (measured as Relative Light Units) for each strain in the presence or
absence of the inducer. You can see our results below (Figure 2).

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 106
Strain A
Strain A + inducer
105
Relative light units (RLU)
Strain B
Strain B + inducer

104

103

102

101
0 6 12 18 24
Hours post-induction
Fig 2. Amount of gene expression in the presence or absence of inducer as measured in
Relative Light Units (RLU) over time.

Experiment 1 Questions

1. How are the plasmids Dr. Richardson’s team used in their experiments similar or different from those you used in your bacterial
transformation investigation(s)?

2. In your own words, describe the purpose of Dr. Richardson’s experiment.

3. What is the advantage of doing experiment 1 before doing an experiment in mice?

4. Which strain (A or B) in the presence or absence of an inducer generated the greatest amount of firefly luciferase as measured
in relative light units?

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Part 3 — In Vivo Experimental Results.
Findings from Experiment 1 demonstrated that the promoters responded differently to the
administered inducer. In a follow-up study not shown here, we determined that the engineered
bacteria were able to survive in the mouse gut. Regulations set forth by the Institutional
Animal Care and Use Committee at our research center were followed in the humane handling of
mice in all of our experiments.

Experiment 2:

The purpose of our second experiment was to determine whether engineered bacteria would
sense infection and stimulate the immune system more rapidly, causing mice to recover more
quickly from dysentery than they would without the bacteria. Our scientists transformed gut-
derived E. coli from mice with a plasmid carrying Promoter A and the immune-stimulating gene
(Strain C) or Promoter B and the immune-stimulating gene (Strain D). Since several different
types of organisms can cause dysentery, we decided to test our engineered strains in
response to bacterial dysentery caused by E. coli O157:H7 or Shigella dysenteriae, or amoebic
dysentery caused by Entamoeba histolytica. The mice were divided into three major groups: those
that received no treatment, those that received engineered bacterial Strain C, or those
that received engineered bacterial Strain D. After three days, the mice were divided into
subgroups each receiving E. coli O157:H7, S. dysenteriae, or E. histolytica to cause dysentery.
After three days of incubation, mice began to show symptoms of dysentery (Figure 3A).
Duration of symptoms in days was tracked for each group (Figure 3B). All mice were provided
IV fluids to prevent dehydration during the course of the trial. The mice were monitored for
a period of 2 weeks. No deaths occurred during the course of the trial.
Infection and Recovery from Dysentery
A

Saline
3 days 1–3 days I.V. s
? days

Dysentery-causing Symptom-free
organisms introduced
Engineered bacteria Infection and
introduced recovery
B
Dysentery-Causing Days to Recovery
Bacteria Introduced Organism Introduced 0 1 2 3 4 5 6 7 8 9

Non-engineered bacteria E. coli O157:H7 7

Strain C, gut-derived E. coli E. coli O157:H7 4

Strain D, gut-derived E. coli E. coli O157:H7 6

Non-engineered bacteria S. dysenteriae 5

Strain C, gut-derived E. coli S. dysenteriae 3

Strain D, gut-derived E. coli S. dysenteriae 6

Non-engineered bacteria E. histolytica 8

Strain C, gut-derived E. coli E. histolytica 7

Strain D, gut-derived E. coli E. histolytica 3

Fig 3. A, overview of dysentery recovery experiment in a mouse model. B, days to recovery

from dysentery symptoms in mice after receiving Strain C or Strain D and a dysentery-causing
organism (E. coli O157:H7, S. dysenteriae, or E. histolytica).

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Experiment 2 Questions

5. What conclusions can Dr. Richardson’s team draw from the data in Figure 3B?

6. What reason can you provide that explains why treatment with engineered bacteria using promoter A and immune-stimulating
gene (Strain C) was different than that using promoter B and immune-stimulating gene (Strain D)?

7. In what ways could this research be used in the future to help humans with dysentery, like those under the care of Dr. Cruz and
Nurse Brady?

8. What considerations might researchers need to make when comparing a mouse system to humans?

Given the results of Dr. Richardson’s data, Dr. Cruz and Nurse Brady felt that these findings demonstrated promise in relieving
dysentery symptoms in mice and could potentially lead to similar treatment of dysentery in humans in the future. This would
be particularly useful in cases such as the one at their military outpost where dysentery was so rampant it overwhelmed the
healthcare facility. However, more research would be necessary in order to fully understand how the engineered bacteria would
work in the human gut.

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9. Consider what the next steps in this work may be in order to better understand how the engineered bacteria work in the mouse
gut to further reduce dysentery symptoms. Provide your ideas for the following:

a. Formulate a follow-up extension question that builds on the research findings presented in this case:

b. Why is your question useful in thinking about the process taking place in the mouse gut?

c. What is your hypothesis? Be sure to provide a reason for your thinking.

d. What data would need to be collected in order to test your hypothesis and answer your question?

e. What protocol steps would you need to take in order to test your hypothesis?

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Bibliography
Aronson NE et al. (2006). In harm’s way: Infections in deployed American military forces. Clin Infect Dis 43, 1045−1051.

Cell Press (2015). MIT scientists hack one of the most common bacteria in the human intestines. AAAS, eurekalert.org/pub_
releases/2015-07/cp-msh070215.php, accessed May 4, 2016.

Forbush D (2016). These four companies are engineering the microbiome to do amazing things. Synbiota, synbiobeta.com/four-
companies-engineering-microbiome-amazing-things/, accessed May 4, 2016.

Kotula JW et al. (2014). Programmable bacteria detect and record an environmental signal in the mammalian gut. PNAS 111,
4838–4843.

Lawrence J (2015). Synthetic biology: Microbes made to manufacture drugs. The Pharmaceutical Journal, pharmaceutical-journal.
com/news-and-analysis/features/synthetic-biology-microbes-made-to-manufacture-drugs/20069575.article#fn_7, accessed May
4, 2016.

Mimee M et al. (2015). Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to
stimuli in the murine gut microbiota. Cell Syst 1, 62–71.

Sanders JW et al. (2005). Military importance of diarrhea: Lessons from the Middle East. Curr Opin in Gastroenterol 21, 9–14.

Wyss Institute (2015). Re-booting the human gut. Wyss Institute, wyss.harvard.edu/viewpressrelease/203/rebooting-the-human-
gut, accessed May 4, 2016.

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