iGEM 2022 — Team ufmg-ufv-brazil

January

### Week 1 (Jan 3 to 7)

We started our first week of the year as an iGEM team! The activities were
initiated by an online meeting when we discussed our previous actions as one
group of synthetic biology studies and started our journey as a team for the iGEM
2022 competition.

Participants: Breno Lisboa, Maira Pena, Laene Abreu,
Livia Galinari, Vanessa Pasa, Yala Sampaio, Gabrielly Mattos, Paulo Muniz

### Week 2 (Jan 10 to 14)

Our team started brainstorming to define a theme for our project.
During this reunion, we also created and discussed a survey integrating each
member's expectations with the progress of our activities until the iGEM competition.

Participants: Breno Lisboa, Maira Pena, Laene Abreu, Vanessa Pasa,
Yala Sampaio, Paulo Muniz, Luiz Penido

### Week 3 (Jan 17 to 21)

We continued our brainstorming ideas in a hybrid meeting.
In addition, we created a term of commitment for the team and
discussed the entry of new members.

Participants: Breno Lisboa, Maira Pena, Laene Abreu,
Vanessa Pasa, Yala Sampaio, Paulo Muniz, Luiz Penido, Livia Galinari

### Week 4 (Jan 24 to 28)

Our team compiled and introduced the main ideas raised to our principal investigators,
Professors Liza and Camila, receiving new insights.

Participants: Breno Lisboa, Camila Souza, Laene Abreu,
Luiz Penido, Vanessa Pasa, Vitor de Morais

February

### Week 1 (Feb 1 to 4)

After debating with our PI Professor Tiago and with the team participants, we could define the theme of our
team's project and start a literature review on the topic of our project!

Participants: Breno Lisboa, Maira Pena, Laene Abreu, Vanessa Pasa, Vitor de Morais,
Yala Sampaio, Paulo Muniz, Luiz Penido, Maria Eduarda Faria, Gabrielly Mattos

### Week 2 (Feb 7 to 11)

We brought our insights from the literature and started to discuss the human practices activities, planning a
collaboration with the "Idea Real" UFMG laboratory to organize a course on biological machines.

Participants: Vitor Marchesan, Maira Pena, Laene Abreu, Vanessa Pasa, Vitor de
Morais,
Yala Sampaio, Paulo Muniz, Luiz Penido, Maria Eduarda Faria, Gabrielly Mattos, Camila Souza

### Week 3 (Feb 14 to 18)

One more week and our literature reviews continued! Besides this, we had a team reunion with our advisors to
present the project and got orientation.

Participants: Maira Pena, Breno Lisboa, Laene Abreu, Livia Galinari, Vanessa Pasa,
Vitor de Morais, Paulo Muniz, Camila Souza, Gabrielly Mattos

### Week 4 (Feb 21 to 25)

By this week, we organized the team's activities and members into laboratory or human practices groups so we
could be more focused. We continued to consult the aspects of the project and discuss an initial workflow and
reagent listing.

Participants: Breno Lisboa, Vitor Marchesan, Maira Pena, Laene Abreu, Vanessa Pasa,
Yala Sampaio, Paulo Muniz, Luiz Penido, Camila Souza

March

### Week 1 (Mar 1 to 4)

We initiated the month in a meeting with our PI Professor Tiago, putting together the principal concepts we
worked on. The team consolidated the project's ideas by beginning the writing.

Participants: Laene Abreu, Vanessa Pasa, Vitor de Morais, Yala Sampaio, Paulo
Muniz,
Luiz Penido, Gabrielly Mattos, Breno Lisboa, Livia Galinari, Camila Souza, Maria Eduarda Faria

### Week 2 (Mar 7 to 11)

The team met with Ali Faraj, a researcher from “Laboratório de Biologia Sintética-UNICAMP” that studies genetic
circuits of metabolic pathways of modified strains of S. cerevisiae. After presenting our project ideas, we
talked
about Assembly techniques. He told us about his experiences and we started to consider using the modular cloning
(MoClo) system for standardized assembly as a strategy for our project. Furthermore, he contributed to the
project
by suggesting the platform Benchling for our constructions. Throughout the week, we researched parts and iGEM
projects involving Lactobacillus sp.

Participants: Maira Pena, Vanessa Pasa, Yala Sampaio, Paulo Muniz, Luiz Penido,
Gabrielly Mattos, Livia Galinari, Camila Souza, Vitor de Morais, Maria Eduarda Faria, Vitor Marchesan, Breno
Lisboa, Laene Abreu

### Week 3 (Mar 14 to 18)

In a meeting with Thaysa Tagliaferri, a post-doctoral researcher at RWTH Aachen University and a participant
from
the UFMG\_Brazil team 2015, we completed our acknowledgment of Assembly methods. She shared her iGEM experiences
and difficulties with BioBrick Assembly. She also shared her experiences with Gibson Assembly and showed us
tools
to design and test primers, such as the NeBuilder platform and OligoAnalyzer. We started to consider using
Gibson
Assembly in our project. She advised us to think a lot about our designs before synthesizing them.

Participants: Livia Galinari, Vanessa Pasa, Laene Abreu, Luiz Penido, Vitor de
Morais,
Yala Sampaio

### Week 4 (Mar 21 to 25)

This week, we talked with Sarah Sacks, a specialist in chitinases, metagenomics, and biocontrol as we had done
a
bioinformatic analysis and were struggling to choose the best protein for our project. We explained to her our
concerns about how to choose the right chitinase and she explained to us the different classifications that are
used for chitinases. Furthermore, she advised us to choose our chitinases considering the optimal pH and
temperature for the activity in the human intestine and suggested using two chitinases together to obtain a
synergetic effect, an endochitinase, and an exochitinase. She also advised us on the usage of signal peptides.
In
addition, we discuss the best way to use the chitinases together: expressing in the same plasmid and same
bacterium or in different plasmids and different bacterium. We continued with the second idea.

Participants: Breno Lisboa, Vanessa Pasa, Maira Pena, Luiz Penido, Maria Eduarda
Faria

### Week 5 (Mar 28 to 31)

This week we prepared a project presentation to show our PI’s. We had a meeting with Professors Liza, Tiago,
and
Camila, where we discussed the experiments we had to do and how we would prove our concept. We also talked about
anthelmintic resistance, the parasites, and their relation with Disability-Adjusted Life Years.

Participants: Livia Galinari, Vitor Marchesan, Laene Abreu, Paulo Muniz, Vanessa
Pasa,
Maira Pena, Luiz Penido, Maria Eduarda Faria, Yala Sampaio, Camila Souza, Vitor de Morais

April

### Week 1 (Apr 4 to 8)

We continued with the weekly general meetings and started having separate lab meetings. We discussed ideas of
kill switches and plasmid design. We thought about obtaining our chitinase gene via RNA extraction from the
fungus
Aspergillus niger, reverse transcription, and PCR with specific primers as a plan B if our synthesized gene did
not arrive on time.

Participants: Luiz Penido, Breno Lisboa, Maira Pena, Livia Galinari, Laene Abreu,
Maria Eduarda Faria, Vitor de Morais, Vanessa Pasa, Yala Sampaio, Paulo Muniz, Camila Souza

### Week 2 (Apr 11 to 15)

We met with Fernando Furtado, a specialist in genetic markers of anthelmintic resistance and neglected tropical
diseases. He explained that the lack of investment was a crucial factor in the lack of development of new drugs
for helminthic diseases. Additionally, he explained the relation between mass drug administration, which is a
control method recommended by the World Health Organization, and the occurrence of anthelmintic resistance.
Furthermore, he emphasized the importance of creating new drugs against these parasites.

Participants: Laene Abreu, Vitor de Morais, Maria Eduarda Faria, Camila Souza,
Vanessa
Pasa

We also had a lab meeting to discuss the next steps of the experimentation.

Participants: Yala Sampaio, Vitor Marchesan, Livia Galinari, Vanessa Pasa, Paulo
Muniz

### Week 3 (Apr 18 to 22)

We had a meeting with our PI, Professor Tiago. He advised us about primer design (to obtain our gene via RNA
extraction) and all the criteria we would need to meet, such as melting temperature. We also discussed the
possibilities of backbones we could use in the project.

Participants: Yala Sampaio, Paulo Muniz, Breno Lisboa, Livia Galinari, Vanessa Pasa,
Vitor Marchesan

On the same day, we met with Fernando Genta, a researcher at the Oswaldo Cruz Institute (FIOCRUZ) in Rio de
Janeiro, who studies chitinases of insect vectors and their relationship with the establishment of parasite
infection, and Maiara do Valle, doctorate by the Stricto Sensu Graduate Program in Parasitic Biology at the
Oswaldo Cruz Institute. We presented our project to them, received insights, and talked about problems that we
could face: low efficiency of secretion and difficulty of the chitinases to access the chitin in the parasites.

Participants: Livia Galinari, Vanessa Pasa, Breno Lisboa

We also met with our PI, Professor Liza, to discuss our new ideas and insights.

Participants: Livia Galinari, Laene Abreu, Maria Eduarda Faria, Vitor de Morais

### Week 4 (Apr 25 to 29)

We had a meeting with our PI, Professor Tiago. We talked about what aspect of our project we would model. He
also
helped us to design primers.

Participants: Livia Galinari, Vanessa Pasa, Breno Lisboa, Maira Pena, Paulo Muniz,
Arthur Enrici

We also had a meeting where we discussed our genetic circuit designs and the creation of our Hardware: a
low-cost
bioreactor.

Participants: Pedro Garcia, Vitor Marchesan, Livia Galinari, Paulo Muniz, Arthur
Enrici, Vanessa Pasa, Luiza Possa, Yala Sampaio, Sávio Mendes, Gabrielly Mattos

May

### Week 1 (May 2 to 6)

We had a lab meeting where we talked about our Hardware, lab experiments, and primer design. At this point we
were thinking about developing 2 Hardware: the low-cost bioreactor and another hardware to mimic the human
intestine, where we could test the activity of our probiotic against worms.

Participants: Pedro Garcia, Vitor Marchesan, Livia Galinari, Vanessa Pasa, Luiza
Possa, Yala Sampaio, Sávio Mendes, Gabrielly Mattos, Breno Lisboa

### Week 2 (May 9 to 13)

We had a meeting with our PI, Professor Tiago. We reviewed the experiment protocols we had gotten from the
literature and received feedback on them. We also showed the PI some comments that Thaysa Tagliaferri had made
on
our primers and adjusted some details on them. Additionally, we discussed Hardware ideas.

Participants: Vanessa Pasa, Paulo Muniz, Vitor Marchesan, Sávio Mendes, Maria
Eduarda
Faria, Yala Sampaio, Gabrielly Mattos

This week, Vitor Marchesan got in touch with a member of UNESP team, that had previously created a hardware to
mimic the human intestine. Vitor, from UNESP, explained the process of developing the hardware and its
difficulties.

### Week 3 (May 16 to 20)

We wrote the protocols we would need to use in the project's next steps and discussed them in a meeting. We
went
through the reagents that we would need. We also planned the next steps with the help of Professor Tiago. In
addition, we sent our primers sequences to synthesize.

Participants: Gabrielly Mattos, Sávio Mendes, Luiza Possa, Livia Galinari, Yala
Sampaio, Paulo Muniz, Maira Pena, Pedro Garcia, Breno Lisboa, Vanessa Pasa

**UFV LAB:**  This week we started to grow the bacteria of interest, *Lactobacillus acidophilus*. To
observe its growth under specific conditions, we cultivated in a plate with solid medium and in Falcons with
liquid medium. With the result of growth, we were able to carry out the storage of the microorganisms in order
to
favor the conduction of experiments in the future.

![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week3-plate.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week3-falcon.png)

Participants: Livia Galinari, Luiza Possa, Arthur Enrici

### Week 4 (May 23 to 31)

This week, we had another meeting with our PI, Professor Tiago, to define the constructions we would synthesize
via IDT grant.

Participants: Livia Galinari, Paulo Muniz, Vanessa Pasa, Maria Eduarda Faria,
Gabriellly Mattos, Breno Lisboa, Sávio Mendes, Luiza Possa, Yala Sampaio

Additionaly, we met to discuss the parameters the bioreactor would need for the cultivation of Lactobacillus
Acidophilus.

Participants: Paulo Muniz, Sávio Mendes, Vitor Marchesan, Vanessa Pasa

**UFV LAB:**  To understand more about *L. acidophilus*, we performed **Susceptibility**  Tests to
observe which antibiotics would be able to inhibit the growth of the bacterium and which concentrations were
necessary for this. For this, we used 4 antibiotics, ampicillin (AMP), chloramphenicol (CLO), kanamycin (KAN)
and erythromycin (ERI), and 3 different concentrations, based on the susceptibility range given by the MIC -
Minimal inhibitory concentration.

![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week4-amp.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week4-clo.png)

![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week4-kan.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/may-week4-ery.png)

Participants:Livia Galinari, Luiza Possa, Arthur Enrici

June

### Week 1 (June 6 to 10)

This week, we had another meeting with our PI, Professor Tiago, to define the constructions we would
synthesize via IDT grant.

Participants: Livia Galinari, Vitor Marchesan, Breno Lisboa, Vanessa Pasa, Luiza
Possa, Sávio Mendes, Pedro Garcia, Paulo Muniz, Yala Sampaio

**UFV LAB:**  This week we focused on the research for protocols and implementation of
**Electrocompetence of L. acidophilus**, in order to prepare the bacteria to receive the plasmid during
transformation. Everything went well and we were able to store the electro-competent in cryo-tubes.

Participants: Livia Galinari, Luiza Possa, Arthur Enrici

### Week 2 (June 13 to 17)

This week, we met to discuss the specific biological parts we would use in our constructions from the IDT
grant. We talked about signal peptides, RBS, promoters and histidine tail.

Participants: Vanessa Pasa, Paulo Muniz, Luiza Possa, Yala Sampaio, Pedro Garcia,
Breno Lisboa, Maria Eduarda Faria

**UFV LAB:**  We decided to perform the transformation in L. acidophilus using one vector with RFP
construct. Although the colonies appeared on the plate, we could not clearly observe them under the
fluorescence microscope. To solve the problem, we tried to grow the bacteria in 100 ug/mL of antibiotic but
there was no growth. However, we observed that the bacteria without the vector grow more than the transformed,
which could inable us to observe the fluorescence.

![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week2-vector.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week2-plate.png)

Participants: Livia Galinari, Luiza Possa, Arthur Enrici

### Week 3 (June 20 to 24)

When we tried to order our constructions from IDT, we had errors and warning messages saying that our
constructions had complexities that prevented the manufacturing of the sequences. We thought about solving
this problem with codon optimization. Finally, we were able to solve this problem by changing the terminators
of our constructions.
We also had a meeting with Professor Tiago to discuss the bioreactor. We talked about the materials that could
be used to reduce costs and the measurement of temperature and pH.

Participants: Yala Sampaio, Livia Galinari, Vanessa Pasa, Vitor Marchesan, Breno
Lisboa, Sávio Mendes, Luiza Possa

**UFV LAB:**  This week we decided to perform the extraction of plasmid DNA, aiming to remove the empty
pExu with the **MiniPrep** technique. After completing the extraction protocols, we decided to assemble an
**agarose gel** to observe the results and quantify how much we obtained during the process. It was seen
that, from 4 extractions, obtained the following plasmid concentrations: 268 ng/ul, 91.4 ng/ul, 177 ng/ul and
152 ng/ul, respectively to the image below.

![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week3-tubes.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week3-gel.png)

Participants: Livia Galinari, Luiza Possa

**UFMG LAB:** After talking to Professor Aristoteles, we obtained permission to perform the experiments
with the fungi in his lab. We cultivated the fungus Aspergillus niger in 4 plates of the solid BDA medium
using the **Protocol of Fungus Cultivation in Solid Medium**. The initial sample was kindly provided by
Paula.

Participants: Vanessa Pasa, Paulo Muniz

### Week 4 (June 27 to July 1)

**UFV LAB:**To finish the month, we decided to transform **L. acidophilus**  with extracted pExu. We
were able to observe the growth of transformed bacteria from our stock of electrocompetent cells. However,
once again we observed that low antibiotic concentrations could be selecting non-transformed with greater
resistance. Thus, we decided that in the coming weeks we would repeat the transformation with higher levels of
antibiotics.

![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week4-pexu.png)

Participants: Livia Galinari, Luiza Possa, Arthur Enrici

**UFMG LAB:** We cultivated the fungus Aspergillus Welwitschia in 2 plates of the Malt Extract Agar using
the **Protocol of Fungus Cultivation in Solid Medium**. The initial sample was kindly provided by Professor
Aristóteles.
We also cultivated the fungus Aspergillus niger for the second time in 2 plates of the BDA medium - from the
samples of the first cultivation - and saw the results of the first cultivation, as shown in the following
pictures:

![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week4-fungus1.jpeg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week4-fungus2.jpeg)

![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week4-fungus3.jpeg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/june-week4-fungus4.jpeg)

One of the BDA plates was contaminated.

Participants: Vanessa Pasa, Yala Sampaio, Paulo Muniz

July

### Week 1 (July 4 to 8)

After discussing for a long time and solving the technical problems, we finally ordered our constructions
from IDT.

**UFV LAB:**  We started the month of July performing the **Growth tests** of *Lactobacillus* for
the
bioreactor. After a group discussion, we came to the conclusion that three main conditions should be tested:
1. Aeration - Growth with presence of Oxygen at 37º C and with rotation of 180 rpm.
2. Incubator - Growth with controlled CO2 levels at 37ºC and without rotation.
3. Anaerobic jar - Growth without oxygen at 37º C and without rotation.
To analyze the growth, we used the spectrophotometry technique at 600 nm at regular intervals for 40 hours.
Our results can be seen in the table below:

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week1-table-new.png )

However, this result confused us a little about Lactobacillus's growth. We expected this species of bacteria
to grow more in environments with no oxygen. Thus, we decided to search the literature for studies that
obtained similar or discrepant results.

Participants: Livia Galianri, Sávio Mendes, Arthur Enrici

**UFMG LAB:**  We transformed E. coli TOP10 using the **E. coli Transformation Protocol** using the iGEM
part BBa\_E0040 because we wanted to miniprep this vector and use it in bio art and in the Biological Machine
Course. We also wanted to test the trasnformation protocol. Unfortunately, it did not work.

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week1-transformation.jpg)

Participants: Yala Sampaio, Luiz Penido, Laene Abreu, Camila
Souza, Maria Eduarda
Faria, Vanessa Pasa

### Week 2 (July 11 to 15)

This week, we received emails from IDT saying that there were problems with our constructions and our order
had been canceled. Vanessa contacted their customer service and they suggested the addition of adapters in the
extremities of our gBlocks. She did these modifications and tried ordering again. This time it finally worked.

**UFMG LAB:**  Unfortunately the flow hood of the laboratory we were using was contaminated. We
decontaminated the flow hood, cleaned it with soap and water, alcohol, and left the UV light on for 4 hours.
We also cultivated Aspergillus Niger using the **Protocol of Fungal Inoculation in Liquid Medium** to test
if the flow hood had been decontaminated, if the fungus grew well even if it was 10 days old and if our liquid
medium worked.

Participants: Gabrielly Mattos, Yala Sampaio, Paulo Muniz, Vanessa Pasa

**UFMG LAB:**  To test our hypothesis of the error of the previous E. coli transformation we performed the
following transformations according to the **E. coli Transformation Protocol**:
- Top10 bacteria + Miniprep RFP (made by Yala in Jan/2020/Ampicillin Resistant).Here, we checked if the
chemically competent TOP10 bacteria was working.
- Bacteria BL21 + RFP Construct BBa\_J04450 (Positive Control/Chlorophenycol Resistant). Here, we checked
whether the positive control of the 2015 iGEM kit was working.
- Bacteria BL21 + BBa\_E0040 (eYFP/Chlorophenycol Resistant). Here, we wanted to miniprep this iGEM vector to
be used in the bio art.
The results were:
The expression of reporter proteins was not observed because we did not put enough IPTG in the plates. So,
using the transformed overnight bacteria left in the Shaker, we re-plated.
It did not work, probably because the growth medium without antibiotics did not have selective pressure to
maintain the bacteria with the plasmid. Plasmid loss.
We transformed E. coli for the third time, using only BL21 + the three plasmids:
- BBa\_E0040 (eYFP)
- RFP (Jan/2020 - Yala)
- Construct RFP BBa\_J04450

It finally worked!

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week2-success.png)

With the troubleshooting of these transformation fails, we learned to use the water bath instead of the dry
bath and to use the right amount of IPTG to observe the fluorescence.

Participants:Yala Sampaio, Vanessa Pasa, Luiz Penido, Laene Abreu, Camila Souza

### Week 3 (July 18 to 22)

We had a lab meeting to discuss the results of our experiments and the next steps.

Participants: Livia Galinari, Sávio Mendes, Gabrielly Mattos, Luiza Possa, Breno
Lisboa, Yala Sampaio

### Week 4 (July 25 to 29)

**UFV LAB:**Due to the problem we had with the transformation results on June 4th, we decided to start
from scratch and extract the pExu plasmid again, but using different protocols.
First of all, we pre-inoculated L. acidophilus pEXU for miniprep using two 50ml falcons, each containing 10 ml
of MRS medium, 10 uL of Erythromycin [50mg/mL] and 10 uL of bacterial stock. After overnight growth, we
obtained 8 aliquots of 2 mL each, of which 4 were frozen for future tests and 4 were used for the miniprep.
For the first extraction, we used lysis by the enzyme lysozyme and continued the experiment using the
**Promega Miniprep Kit and Plasmid Extraction**.

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week4-bluetubes.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week4-tubes.png)

For the other two samples, we used standard extraction protocols for Lactobacillus lactis and Escherichia
coli. In this way, we were able to analyze the best way to extract the Lactobacillus acidophilus plasmid.

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week4-gel.png)

After all the work, we proved the efficient extraction of plasmid DNA from the bacteria through a 1% agarose
gel.

Participants:Luiza Possa, Livia Galinari

**UFMG LAB:** We did the **Protocol of Chemically Competent Cell Preparation** with E. coli BL21 to
transform it with GFP and RFP (E. coli Transformation Protocol) and use it in the bioart activities from IV
Engineering Course of Biological Machines. It worked as expected.

![](https://static.igem.wiki/teams/4349/wiki/notebook/july-week4-rfp.jpeg)

August

### Week 1 (Aug 1 to 5)

We met with Professor Tiago to discuss the lab activities and start the modeling process. Our team did not
have previous knowledge of mathematical modeling, so it was hard to start this activity. We discussed the
best software and the variables we would incorporate into the model.

Participants: Vanessa Pasa, Livia Galinari, Sávio Mendes, Breno Lisboa,
Gabrielly Mattos, Yala Sampaio, Luiza Possa, Paulo Muniz

**UFV LAB:** During the month of August, we, from the UFV laboratory, decided to take some time off the
bench
and go home. In this time, we took the opportunity to write for the Wiki and to calculate our route for the
next experiments.

**UFMG LAB:** Our IDT construction arrived in the lab. We ressuspended the IDT gBlocks in water and stocked them in the freezer (-20C). Furthermore, we prepared
agar plates to be used in the IV Engineering Course of Biological Machines with the Protocol for Preparation
of Agar Plates.

Participants: Yala Sampaio, Vanessa Pasa, Paulo Muniz, Luiz Penido, Camila
Souza, Maria Eduarda Faria

### Week 2 (Aug 8 to 12)

This week we had an event in UFMG, the VI Engineering Course of Biological Machines. The lab team members
helped in the organization of the course, being monitors. Professor Tiago also came from UFV to participate
as a lecturer.
We also had a meeting with our advisors and received orientation.

Participants: Yala Sampaio, Livia Galinari, Maira Pena, Maria Eduarda Faria,
Vanessa Pasa, Breno Lisboa, Gabrielly Mattos, Laene Abreu, Luiza Possa, Luiz Penido, Sávio Mendes, Vitor de
Morais

### Week 3 (Aug 15 to 19)

We started the modeling activities in RStudio. At this point, we had obtained all the variables needed from
the literature and we started coding. Our model would be divided into 3 steps:
1- Determining the viable Lactobacillus acidophilus cells that would be able to arrive in the human
intestine according to the initial dose.
2- Determining the amount of chitinase produced by the cells.
3- Determining the anthelmintic activity. Determining how many helminth units the probiotic doses we
speculated would be able to kill/eliminate.

**UFMG Lab:** First, we performed the addition of adenine overhangs in the constructs from IDT, using the
**Tailing for TA Cloning Protocol**, for insertion in pGEM T Easy Vector via TA cloning. We used 100ng of
each gBlock - endochitinase and exochitinase. Thus, we used 10uL of our initial stock of the two enzymes.

We realized that the final result of the adenine addition protocol was in very low concentration, that is,
our gBlocks were very diluted. We could not achieve the wanted ligation ratio of 1:3 (vector: insert)
because of this dilution and we did not know how to solve this problem. We tried to ligate with a lower
insert ratio and proceeded with the transformation and Blue/White Screening of E.coli cloning strains TOP10
and DH5α.

The results are shown in the picture:

![](https://static.igem.wiki/teams/4349/wiki/notebook/august-week3-plates.jpeg)

There was no colony growth using TOP10. We stopped using this strain because it was probably not chemically
competent anymore because it was old. DH5α had growth of few blue colonies, so the ligation and transformation
were not efficient and the pGEM T Easy vector closed on itself.

Participants: Yala Sampaio, Vanessa Pasa, Paulo Muniz

### Week 4 (Aug 22 to 26)

We continued with the modeling activities and were able to plot graphs that correlated the time with the
viable Lactobacillus acidophilus cells in the human intestine.

**UFMG Lab:**This week we faced an unfortunate problem in the lab. We arrived at the lab and found out
that the freezer where we were storing our samples had been shut down for at least 2 days (weekend). We were
worried that we would lose everything, but we had to keep going.
Because we were using DH5α aliquots lent from another lab, we decided to cultivate them and make a stock of
DH5α competent cells for our lab. We proceeded with the **Protocol of Chemically Competent Cell
Preparation**. Then, we tested the competence of our cells by transforming them with a pUC vector and
RFP. There was no colony growth.
To understand what was happening and to test the relation between bacteria strains and vectors, we
transformed all the possible combinations between the bacteria: DH5α, BL21, TOP10; and the vectors: RFPcemb,
RFPnew, pETL, pUC.

The results were:

Growth only in (DH5α + RFPnew), (BL21 + RFPnew), (DH5 + RFPcemb), (TOP10 + RFPcemb) and (TOP10 + pETL).

Participants: Yala Sampaio, Vanessa Pasa, Gabrielly Mattos, Paulo Muniz

### Week 5 (Aug 29 to Sep 2)

**UFV Lab:** We ended the month of August meeting after the holidays. At this point, we discussed the lab
part
together and tried to outline our next steps. It was decided that we would follow the following order of
procedures:
1. TA cloning
2. Transformation of Escherichia coli DH5 alpha by heat shock.
3. Selection of the best bacterial colonies for growth in liquid medium.
4. Miniprep and agarose gel to confirm insertion.

Participants: Luiza Possa, Lívia Galinari, Arthur Enrici

**UFMG LAB:** We did not understand the divergence of the transformation results. We thought that they could
have been impacted by the usage of old agar plates. We transformed E.coli again to see if the old agar plates
were
problem.

We plated the 4 combinations between (BL21, DH5α) and (pETL, RFPnew) in old and newly made agar plates for
comparison. We were unable to achieve a conclusion
because there was growth in old and new plates, as shown in the picture below:

![](https://static.igem.wiki/teams/4349/wiki/notebook/week5-aug.png)

However, we decided to use only new agar plates in the next experiments to prevent any problems related to
that.

Then, we tested if our pGEM-T Easy Vector was still working after the freezer incident. We performed the
ligation
without the insert to try to achieve blue colonies. We followed the **NEB Ligation Protocol**, then
transformed
E. coli and did not obtain colony growth.

Participants:Yala Sampaio, Vanessa Pasa, Camila Souza

September

### Week 1 (Sep 5 to 9)

We had another meeting with Professor Tiago to discuss the modeling problems and difficulties. We did a linear
regression to obtain the relation between the probiotic factor and time. However, we later discovered that a
polynomial function was not the best function to describe this relation.

Participants: Vanessa Pasa, Vitor Marchesan, Livia Galinari

**UFV LAB:** This week, after receiving the UFMG sample of 10 gblocks, we decided to carry out the **tailing
experiment**. This technique is an enzymatic method for adding an untemplated nucleotide to the 3' end of a
double-stranded DNA molecule. Thus, we started to prepare our T-vector for use in TA cloning.

The next day, we continued with the **TA cloning**. To perform a 20 uL reaction, we used 5 uL of gblock (we
first
tested two samples at random to see if the protocol would work: Sec 1 and Mel2), 0.5 uL of Taq, 2 uL of buffer,
1
uL of dATP and 11.5 uL of ultra pure nuclease free H2O.

This aliquots were then incubated at 70°C for 30 minutes. After this time, the samples were placed to dry for
one
hour in an aqueous vacuum centrifuge, maintaining the temperature of 30°C.
After the material had completely dried, we made the ligation with pGEM T easy, using the **Promega Protocol**.

With the last experiment done, we now needed to perform the **DH5 alpha transformation**. For this, we used the
thermal shock technique, following the following steps: first, we placed 3 uL of the binding reaction and left
it
on ice for 30 minutes. After the time had elapsed, the tubes were heated at 42°C for 40 seconds and then left on
ice for 2 minutes.

The samples were then centrifuged at 5000 rpm for 5 minutes. After separation, we collected uL of the
supernatant
and resuspended the colonies.

The samples were then centrifuged at 5000 rpm for 5 minutes. After separation, we collected uL of the
supernatant
and resuspended the colonies to then be plated in Petri dishes containing 100 ug/mL of AMP and X-gal, and IPTG.
Incubation was carried out in an oven for 24 hours at 37°C.

As can be seen in the images below, the results show many blue colonies and satellite colonies, with a few
isolated white ones.

![](https://static.igem.wiki/teams/4349/wiki/notebook/sep1-plate1.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/sep1-plate2.png)

We passed the white colony of Sec1 (not isolated) to 5 mL of liquid LB antibiotic medium (AMP 100ug/mL) and wu
choosed a random white colony of Mel2 to try.

Unfortunately, only Sec1 grew in the antibiotic liquid medium. As it was not possible to get isolated colony on
a
plate, we decided to make a streak with the liquid medium. In addition, we tried to verify in the fluorescence
microscope the presence of signs indicating that the transformation occurred correctly. However, no fluorescence
in GFP was observed.

![](https://static.igem.wiki/teams/4349/wiki/notebook/sep1-ufv.png)

Participants: Lívia Galinari, Luiza Possa, Sávio Mendes, Arthur
Enrici

**UFMG LAB:** We decided to start from the beginning again, with another cloning vector, which had not been
thawed in the freezer incident: TOPO. The adenine overhang was added using the **Tailing for TA Cloning
protocol.** We purposely used more insert, because we feared that the DNA had been degraded because of the
shutdown of the freezer. Then, we concentrated the samples using SpeedVac. After that, we proceeded with the
insertion of our A-tailed gBlocks in the vector, using the **Cloning with TOPO Protocol**. We used the bacteria
DH5α and BL21 to perform the transformation.
We expected “carpet growth” on the plates, but unfortunately few colonies (and small) grew. Thanks to Luciana, a
PhD of UFMG Lab, we were able to see the tiny colonies! We were so happy that it finally worked!

![](https://static.igem.wiki/teams/4349/wiki/notebook/suc1.jpg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/suc2.jpg)

![](https://static.igem.wiki/teams/4349/wiki/notebook/suc3.jpg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/suc4.jpg)

With the help of pipette tips, we took beautiful colonies (which did not have satellites) and threw them in the
liquid medium with antibiotic. We let them grow overnight in the Shaker in 4 ml LB medium. We also replated the
colonies in a new agar plate with antibiotic, to have another “backup”, as shown in the picture:

![](https://static.igem.wiki/teams/4349/wiki/notebook/estoque.jpeg)

In the next day, we observed that the Liquid Inoculum and the agar plate results were consistent with each
other.
If the bacteria grew in the plate, it also grew in the falcon. The results are shown in the pictures below:

![](https://static.igem.wiki/teams/4349/wiki/notebook/estoque-2.jpg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/estoque3.jpg)

![](https://static.igem.wiki/teams/4349/wiki/notebook/estoque4.jpg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/estoque5.jpg)

Of the samples that grew successfully in the liquid medium, 2 aliquots were stored with 200 ul each (100ul
of
bacteria + 100 ul glycerol 50%), according to the Protocol of Bacterial Glycerol Stock. Stored at -80ºC!

Participants: Yala Sampaio

Then, we did a Miniprep of the samples according to the **Miniprep Nucleo Spin Plasmid Protocol.**
Unfortunately, we forgot to add RNAse to Buffer A1, because we discovered later that it should have been
added.
After that, we digested our plasmids with EcoRI according to **EcoRI Digestion Protocol**

To confirm the digestions and the insertion of our gBlocks in the TOPO vector, we proceeded with gel
electrophoresis using the Gel Electrophoresis Protocol. We used 1% agarose gel of 50 ml. We used the comb with
14
wells. The result was:

![](https://static.igem.wiki/teams/4349/wiki/notebook/geleco.png)

All samples showed the bands as expected, the only problem was the blur at the bottom due to RNA
contamination
(we forgot to use RNAse in the Miniprep), but it did not compromise the result. We can confirm with the
virtual
digestion:

![](https://static.igem.wiki/teams/4349/wiki/notebook/virtual-digest-sequence-pcr21-topoendo.png)

Participants: Yala Sampaio, Vanessa Pasa

### Week 2 (Sep 12 to 16)

**UFV LAB:**In the second week of the month, we performed the **tailing experiment** again, as we
judged the
results of the previous one to be inconclusive. This time, we redid the process using 15 uL of gblock, that
is,
150 ng. The next day we carried out the transformation.
After 24 hours, as seen below, the colonies grew!!!

![](https://static.igem.wiki/teams/4349/wiki/notebook/week2-sep.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/novafoto.png)

In this way, we collected the isolated white colonies and transferred them to the liquid medium. However,
unfortunately, they showed no growth after 24 hours.

To find out if the colonies were still viable, we put them in a Petri dish, separating them into small
squares.
In addition, we performed a negative control with untransformed L. acidophilus to compare the growth.

![](https://static.igem.wiki/teams/4349/wiki/notebook/quadrinhos.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/quadrinhos2.png)

Participants: Lívia Galinari, Sávio Mendes, Luiza Possa

In the same week, we decided to perform the Lactobacillus acidophilus electrocompetence. However, for making
stocks, we double the protocol. Therefore, we started by performing the pre-inoculum in 30 mL of liquid medium
using 10 uL of the bacterial stock. The following day, the 30 ml were diluted in 200 ml of 1% glycine 20%
(v/v)
medium.
After a tiring 7 hours, the inoculum reached an OD of 0.550 and we started the electrocompetence protocol. As
a
result, we obtained 20 tubes with 100 uL each that were stored at -80°C.

Participants: Lívia Galinari, Arthur Wakim

To finish this week of hard work, we tried to carry out tests with the vectors. For this, we collected the
previously inoculated colonies on the plate with the quadrants and passed them separately to 3 mL of liquid LB
medium with AMP. The falcons were then left at 37°C for 24 hours at 180 rpm. However, unfortunately, nothing
grew
back.
We looked for Professor Tiago so that he could help us with this issue. Therefore, he asked us to run the
tests
using another pGEM and performing positive and negative controls.

Participants: Luiza Possa

**UFMG LAB:**Our next goal was to insert our constructions into an iGEM expression vector, to be able to
express our proteins. We decided to try to use RFP construct and pSB1C3 as our vectors (backbones).
We proceeded with the digestion with EcoRI of the linearized plasmid Backbone pSB1C3 (25ng/ul), Exochitinase
4X
(Nanodrop 615,9ng/ul on 8/9/22), Endochitinase NA1 (Nanodrop 425,6ng/ul on 8/9/22) and Miniprep of the plasmid
RFP. We selected these Exochitinase and Endochitinase because they showed stronger bands in the previous gel
and
had no other bands.
Digestion was performed according to **EcoRI Digestion Protocol** and we made another 1% agarose gel
electrophoresis using the **Gel Electrophoresis Protocol**. Here is the result:

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-12-08-22.png)

We noticed that the Miniprep RFP did not work in the gel, so we used pSB1C3 as our expression vector. After
that,
we performed the extraction of the appropriate (digested) bands and followed the Protocol for DNA purification
of
the gel bands. We eluted the final samples with 15uL. Measure of the digested samples in the Nanodrop:

EXO 4X = 9.2ng/ul

ENDO NA1 = 4.5ng/ul

pSB1C3 = 6ng/ul>

To proceed with the insertion of our genes of interest in the pSB1C, we digested the 3 resultant samples with
**PstI digestion Protocol**. Then, we precipitated the post-digestion DNA samples with the **DNA Precipitation
Protocol.** After that, we ligated the backbone + the inserts with the **T4 DNA Ligase Protocol** and left the
reaction in the Thermocycler 16°C overnight.

Then, we transformed BL21 bacteria with the pSB1C3 vector with our exochitinase and endochitinase inserts
with
the **E. coli Transformation Protocol**.

![](https://static.igem.wiki/teams/4349/wiki/notebook/endo-bl21-100ul.png)

![](https://static.igem.wiki/teams/4349/wiki/notebook/endo-bl21-200ul.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/exo-bl21-100ul.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/exo-bl21-200ul.png)

It worked! We were able to successfully transform our bacteria to express exochitinase and endochitinase!

Participants: Yala Sampaio, Vanessa Pasa

Using pipette tips, Yala placed each colony in 4ml of LB + 2ul of Chloramphenicol (50ng/ul) in falcons that
she
left in the shaker overnight.

With another tips she did striation of the colonies on a new agar plate with chloramphenicol (this plate was
used
as another stock!) and left the agar in the incubator overnight.

![](https://static.igem.wiki/teams/4349/wiki/notebook/stock.jpg)

All colonies grew on the agar plates and in the liquid medium. So we have this plate as backup!

Yala also did Bacterial Glycerol Stock (50ul bacteria + 50ul glycerol) with the colonies that
grew in the liquid medium. She stored the stock in -80C.

She did the **Miniprep Protocol**, this time, adding RNAse. She measured the samples in
the
Nanodrop, with the following results:

To guarantee that the colonies that grew had our inserts we digested the miniprep with NotI, using the
Protocol
of NotI Digestion. Then, we did another gel electrophoresis 1%.

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-confirm.png)

With the gel we concluded that our inserts were in the pSB1C3 vector and in our BL21 bacteria!

Participants: Yala Sampaio, Vanessa Pasa

### Week 3 (Sep 19 to 23)

**UFV LAB:**To verify the presence of the inserted vector in E.coli, we initially performed miniprep and
EcoRI
digestion. For miniprep, the Promega Kit and protocols were used again. The quantification in the Sec1 qubit
was
424 ng/uL.
For digestion, it was successfully performed. The appearance of equivalent bands was observed, proving the
insertion of the vector in the plasmid.

Participants: Luiza Possa, Lívia Galinari

As our last attempts to ligate our inserts to pGEM T easy were unsuccessful, we tried one last time to carry
out the process and transform using the same protocols. This time many blue colonies appeared, however few
isolated white colonies could be seen.

![](https://static.igem.wiki/teams/4349/wiki/notebook/f2.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/f3.png)

![](https://static.igem.wiki/teams/4349/wiki/notebook/f5.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/f6.png)

**UFMG Lab:** This week, we studied our next steps.

Participants: Yala Sampaio, Vanessa Pasa, Paulo Muniz

### Week 4 (Sep 26 to 30)

**UFV LAB:**During this week some white colonies that appeared to be isolated were selected and inoculated
into Petri dishes containing AMP, X-gal and IPTG.
The colonies that grew were transferred to 15 mL falcon tubes containing 3 mL of antibiotic liquid medium and
left
in the shaker at 37°C and 180 rpm overnight.
Unfortunately, only one culture appeared to grow in liquid medium. However, when observing the fluorescence
microscope, contamination was identified.

Participants: Livia Galinari, Luiza Possa, Arthur Enrici

As our Kit didn't arrive in time, we needed the help of the Unicamp team so that they could lend us the psB1
vector to put in the sequences. They sent the BBa\_J04450 part, however it contains a RFP coding region and we
only
needed the empty pSB1C3 vector. In this way, we digested with restriction enzymes and performed a simulation
in
Benchling to know which band of the gel would have the empty psB1C3 (backbone). To know which buffer would be
the
most ideal for both enzymes, we tested 3 buffers: Multicore buffer (Promega), O buffer (Thermo Fisher) and H
buffer (Sigma).
According to the Benchling digestion, we saw that the backbone would be in the upper band and the results of
the
digestion of the sequence Bba\_J04450 with EcoRI and Pstl can be seen below.

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-backbone1.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-backbone2.png)

Then, we cutted the upper band of the gel and purified using BioFlux Gel Extraction Kit.

**UFMG LAB:** This week, we confirmed the insertion of our construction in the plasmid pSB1C3 using PCR and Vf and Vr
primers.
![](https://static.igem.wiki/teams/4349/wiki/notebook/pcr.png)

We also prepared a small expression for both enzymes. Assuming that our enzymes are exported to the
extracellular space because of the addition of the signal peptide to our constructs, we performed nickel
column affinity chromatography to purify our enzymes with histidine-tail.

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-30-09-22.png)

We use polyacrylamide gel to determine the presence of our enzymes in the supernatant and eluate.
Unfortunately, it was not possible to observe any band.

Participants: Vanessa Pasa, Yala Sampaio

October

### Week 1 (Oct 3 to 7)

**UFMG LAB:**In search of investigating the expression of our enzyme,we cultured 1 liter of each enzyme in
LB, and separated its supernatant and pellet. Afterward, we concentrated our supernatant, as in theory our
enzymes should be secreted. We then sonicated the pellet, separating the soluble fraction and the insoluble
fraction, to cover all our possibilities. Then, we made a new polyacrylamide gel.

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-05-10-22.png)

Unfortunately, it was not possible to add the pellet and insoluble fraction samples as they would float and
enter the gel.

Participants: Vanessa Pasa, Yala Sampaio

**UFV LAB:** since we couldn't clone the fragments into a cloning vector, the group on UFMG lab sent us a
sample of the chitinases (optimized for  *E. coli* ) cloned into pSB1C3 vector. So we transformed them into
 *E. coli*  via thermic chock. The bacteria grew on selective plate and we confirmed the transformation
with a colony PCR, using as a positive control the BBa\_J04450 miniprep. We tested two sets of VF VR primers to
see if there was any contamination in the samples. As shown in the gel, Exochitinase has a fragment around
1500bp, bigger than Endochitinase, which is observed around 1200bp.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-colony-pcr-e-coli-ufv.png)

Then we transformed these fragments into  *L. acidophilus* , they grew on the plate and then in MRS broth
supplemented with 50 ug/mL of Cloramphenicol. We checked the bacteria in the microscopy for contamination and it
was not contaminated.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lac-exo-ufv.jpeg)
![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lac-endoufv.jpeg)

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lac-clo-ufv.png)

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lacmmicrosc-ufv.png)

Then we performed a colony PCR using 10 minutes in the hot start, but our samples did not showed any bands,
maybe because DNA of Gram-positive bacteria is harder to exctract. So we did a miniprep and then PCR, which
showed a lot of unespecific bands and we would need to repeat it.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-colony-pcr-lac-ufv.png)
![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-pcr-lac-ufv.png)

And in our final attempt to confirm the transformation in  *L. acidophilus*  we tried a protocol to
convert chitin, which is highly insoluble, in colloidal chitin. Then we prepared MRS-agar medium with 2% of
colloidal chitin, poured in on the plates and made holes to apply 5 uL of bacteria grown overnight. It seems to
appear a degradation halo around two of the chitinases showed in the image. This may be a sign of chitin
degradation by the chitinases, but we also would need more testing to assure it.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-halo-lac-ufv.png)

Participants: Livia Galinari, Luiza Possa, Arthur Enrici, Sávio Mendes

# Engineering Success

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img3.jpg)

---

# Interactive development of the hardware

## Reactor Vessel

As a first step, we scrutinized that common lab glassware was our best option for the bioreactor vessel.
 Common lab glassware meets a few criteria: it is standardized, has high transparency, it is autoclavable, cheap, easy to buy, and you may already have it in your lab. So,  **the chosen vessel was a 1L Lab bottle with GLS-45 mouth (small mouth)** and we simulated it on a 3D Computer Aided Design Software (CAD), as shown below.

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img1.png)

*Fig 1. 3D CAD illustration of 1L GLS-45 as a reactor*

Yet, we noticed that thin pH sensors are not common nor are cheap. So, we tried this glassware with a benchtop pH sensor, and realized it wouldn’t fit many things beside the sensor, also the cap would be fragile with too many holes and thin spacing. **To solve this, we changed to the same glassware, but with a wider mouth: 1L GLS-80 (wide mouth).**

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img2.png)

*Fig 2. 3D CAD illustration of 1L GLS-80 as a reactor*

---

## Reactor Lid

##### FIRST CYCLE ROUND

At first, we considered a commercial lid with 5 multiple input-outputs (as shown above), nonetheless, this is too expensive and may not fit all tubing and sensors, reducing flexibility. So we considered designing a lid to be 3D printed using additive deposition manufacturing, yet available plastics for this technique doesn’t handle well autoclave, nor have good sealing between printed layers. Our next option was to build the top part using other material that can stand autoclave, is nontoxic, is resistant to acid and basic agents, and holds the form. The material that suits it all and it is not expensive nor require many resources to create is the **polymerizable silicon rubber.**

Yet, the platinum silicone was out of stock in our country due to pandemics and probably to high demand in medical-industrial sector. To circumvent, we migrated to **non-platinum silicone rubber**, that contains a small quantity of components that may interact with cultured cells. Nonetheless, due the minimal exposure of the lid to the culture medium, the risk was accepted. We have chosen a silicone rubber that is capable to withstand up to 250°C and is red colored.

We first designed a shaping mold on 3D CAD software and printed it:

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img3.jpg)

*Fig 3. 3D CAD design*

But results weren’t good: one plastic pin broke due to bad printing configuration, the silicone leaked due to inadequate connection design, and the lid wall had almost no contact with the glass.

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img4.png)

*Fig 4. First silicone iteration*

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/gif1.gif)

##### SECOND CYCLE ROUND

So, we made a new mold with inside walls more conic shaped, we designed a better wall junction, and printed again.

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img5.png)

*Fig 5. Second lid iteration*

Most problems were solved, and we decided it was time to our first test with living organisms. But again, a new problem appeared: when testing the lid, we noticed it started to crack. Our supposition was that the proportion of silicon to catalyzer was incorrect and a new attempt with the same mold was made.

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/img6.png)

*Fig 6. First and second attempt with lid mold version 2*

As the material was as brittle in the first attempt, we discovered that the rubber was too hard to this function! So, we searched again for a new rubber, with hardness bellow the red silicone, which has a hardness grade of shore A 45. We were not able to find any rubber in stock that has a hardness bellow shore A 45 and was able to stand up to 120°C of an autoclave cycle.
![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/gif2.gif)

##### THIRD CYCLE ROUND

One silicone supplier informed that one of his silicones, a **white rubber with shore A 10** , probably would handle the autoclave cycle, and we tried it. And finally, we could test with a living organism.

Also, on this first test, we planned to use a syringe directly connected to the tubbing, but we soon discovered this wasn’t the best scenario as it was hard to attach the syringe nozzle to the tube, and it scaped twice of it too. On the second attempt with the white rubber, we also created a sealed sampling port using the same white rubber and pipette tips.

![](https://static.igem.wiki/teams/4349/wiki/enginer/image7.png)

*Fig 7. First and second attempt with white silicone rubber, and a new tool created for the reactor, a sealed sampling port using pipette tips and silicone rubber.*

Meanwhile, we worked on the mold too. As we chose to work with standard pH sensor to avoid the high-cost long pH sensors, we found that the minimum working volume was 800mL in a 1000mL flask. As we noted that there were room on the rubber lid, we designed a simple attachment that enabled us to move the pH sensor 2.5 cm down on the flask.

![](https://static.igem.wiki/teams/4349/wiki/enginer/image8.jpeg)

*Fig 8. A simple mod was developed to the mold, so we gained 2,5cm of pH movement, and reduced the minimum working volume from 800mL to 700mL.*

Yet, the white rubber became softer and sticky after just a few trials, it started to degrade due to autoclave cycles.

![](https://static.igem.wiki/teams/4349/wiki/enginer/image9.png)

*Fig 9. Picture showing white silicone degradation after some cycles of use.*

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/gif3.gif)

##### FOURTH CYCLE ROUND

A new solution was needed. We simply tested another silicone with hardness shore A 15 green colored, besides the datasheet describing a maximum temperature of 80°C.

**The green silicone worked better then expected, with no signs of degradation even after more than 6 cycles.**

![](https://static.igem.wiki/teams/4349/wiki/enginer/img-10.png)

*Fig 10. The third silicone rubber material tested.*

![](https://static.igem.wiki/teams/4349/wiki/enginer/enginer/gif4.gif)

---

##### Additional information about hardware design can be found [HERE.](https://2022.igem.wiki/ufmg-ufv-brazil/hardware)

---

Chitinase Docking Simulation

In order to evaluate if our proteins would bind correctly to a chitin polymer, we performed a molecular docking
analysis. We retrived a structure for the chitin octamer in PubChem
(https://pubchem.ncbi.nlm.nih.gov/compound/Chitin-Octamer).
For the chitinases, we searched their entry in the UniProt databank and retrived their structure available from
AlphaFold: A0A179G878 for Pcchi44 and G3Y2N5 for CfcI.
With the structures in hand, we performed the molecular docking simulation with SwissDock[1]. After retrieving the
docked structures, we calculated the binding free energy with PRODIGY[2].

[1] GROSDIDIER, Aurelien; ZOETE, Vincent; MICHIELIN, Olivier. SwissDock, a protein-small molecule docking web
service based on EADock DSS. Nucleic acids research, v. 39, n. suppl\_2, p. W270-W277, 2011.

[2] XUE, Li C. et al. PRODIGY: a web server for predicting the binding affinity of protein–protein complexes.
Bioinformatics, v. 32, n. 23, p. 3676-3678, 2016.

E. coli Transformation Protocol

### Materials

* Ice bath
* Incubator
* Aliquot of chemically competent E. coli bacteria
* Plasmid
* Liquid LB medium
* Solid LB medium with antibiotic
* Pipettes and tips
* Shaker
* Water bath
* 1,5ml microtubes
* Laminar flow hood

### Methodology

1. Remove the bacteria from the -80ºC freezer and thaw on ice;
2. Turn on the water bath at 42ºC;
3. Thaw DNA plasmid on ice;
4. Add 1ul of DNA at the desired concentration to the tube containing the chemically competent bacteria;
5. Incubate the tube on ice for 30 min;
6. Heat shock in the bath at 42ºC for 30s;
7. Incubate immediately on ice for 2 min;
8. Add to the tube 600ul of LB liquid medium gently;
9. Incubate the tube at 37ºC under agitation in the shaker (~120rpm) for 1h30min;
10. Remove an aliquot (suggested: 200ul) and plate (Solid LB + antibiotic);
11. Incubate the plates with the bacteria overnight in the incubator at 37ºC;
12. Wait 16h and see if the bacteria have grown.

Protocol of Chemically Competent Cell Preparation for E. coli

### Materials

* Eppendorf tubes
* Falcon
* Ice bath
* Aliquot of E. coli bacteria
* Pipettes and tips
* Shaker
* Solutions: a)100mM CaCl2; b)100mM CaCl2 with 20% glycerol; LB;
* Laminar flow hood

### Methodology

**Day 01**

1. Inoculate 50ul of the desired bacteria in 5ml of medium LB in a Falcon;
2. Leave the falcon overnight in the shaker at 37ºC;

**Day 02**

1. Transfer 1ml of pre-inoculum to 50ml of LB into an erlen;
2. Grow cells up to 5x10^7 (OD 0.4 - 0.5). Usually it takes around 1-2 hrs;
3. Incubate on ice for 10 min. From now on, everything must be done on ice;
4. Heat shock in the bath at 42ºC for 30s;
5. Incubate immediately on ice for 2 min;
6. Centrifuge at 4ºC, 4,000g for 8 min;
7. Discard the supernatant;
8. Resuspend in 25ml (1/2 of LB volume) with 100mM CaCl2;
9. Leave on ice for 5 min;
10. Centrifuge at 4ºC, 4,000g for 8 min;
11. Discard the supernatant;
12. Resuspend in 2ml (1/25 of LB volume) with 100mM of CaCl2+Glycerol;
13. Keep in refrigerator 4ºC for 12-24hrs;

**Day 03**

1. Dispense 50ul-200ul in Eppendorf tubes;
2. Store at -80ºC immediately;

Tailing for TA Cloning Protocol

### Methodology

The tailing procedure consists of adding a single adenine overhang on the 3’ ends, to make the gene fragment
compatible with TA cloning.

1. Add the following reagents to a 20uL reaction:

* Gene fragment: 50ng* Taq polymerase: 1-3 units* 10x Taq polymerase buffer (-Mg): 2ul* 1mM dATP: to [0.2mM]* 25mM MgCl2: to [1.5mM]* Nuclease-free water: to final 20ul volume

2. Incubate at 70ºC for 30 minutes;

If necessary, it is possible to add more quantity of Gene fragment. After the shutdown of the freezer we
decided to add more gene fragment because we were scared our DNA could have been degraded.
This protocol was taken from the document: “TA cloning method” from IDT[1].

**References**

[1]gBlocks and gBlocks HiFi Gene Fragments | IDT. (n.d.). Integrated DNA Technologies. Retrieved September 21,
2022, from
https://www.idtdna.com/pages/products/genes-and-gene-fragments/double-stranded-dna-fragments/gblocks-gene-fragments

#### Contents

* [Wet Lab](#Wet Lab)
* [Docking](#Docking)
* [Modelling](#Modelling)
* [Hardware](#Hardware)

![](https://static.igem.wiki/teams/4349/wiki/capa-results1.jpeg)

# Overview

We’ve achieved results mainly in four segments: wet lab, modelling, docking, and hardware.

# Wet Lab

Our plans at the wet lab consisted mainly of three parts. First, the expression of our enzyme using
*E.
coli* as a chassis. Secondly, expressing our enzyme at *Lactobacillus acidophilus* as a
probiotic.
And last but not least, testing our enzymes on *C. elegans*, a nematode model organism. As
demonstrated
in the image below, the tasks were divided between the two universities of the team being UFMG in charge
of
the expression and characterization of both chitinases in *E. coli* and the test of nematicidal
effect.
And UFV responsible for characterizing various signal peptides designed and quantifying the production of
the chitinases.

![](https://static.igem.wiki/teams/4349/wiki/results/results-plans2.png)

## Expression of Endo and Exochitinase in *E. coli*

Although our objective is to express endo and exochitinase in *Lactobacillus*, at first, we
developed
a composite part for expression in *E. coli*, as it is a well-studied and characterized model
organism.

**Design**

For this, our team designed a composite part to express and secrete CfcI exochitinase from
*Aspergillus
niger* and PCCHI44 endochitinase from *Pochonia chlamydosporia* in *E. coli*. Both
constructs
consist of a strong constitutive promoter, RBS, NSP4 signal peptide, CDS, histidine tag (6x) and
terminator.

![](https://static.igem.wiki/teams/4349/wiki/results/circuito-endochi-e-coli-bba-k4349007.png)

![](https://static.igem.wiki/teams/4349/wiki/results/circuito-exochi-ecoli.png)

*Fig 1. Endochi and exochi construction parts*

We requested both composite parts of the IDT Grant flanked with restriction enzyme site EcoRI and XBal
in
the prefix and Spel and PstI in the suffix to facilitate our future digestion.

**Cloning**

We added 3' adenine overhangs with Taq polymerase and proceeded to TA cloning, inserting the construct
in
pCR™2.1-TOPO™ cloning vector from ThermoFisher (the vector comes with thymine overhangs). Then, we
transformed DH5alpha E.coli with the resultant plasmid.

![](http://parts.igem.org/wiki/images/9/93/DH5ALPHA-ENDO-200UL.jpg)

Fig 2. Transformed DH5alpha with pCR™2.1-TOPO™ cloning vector +
BBa\_K4349007 construct. Colonies are shown inside the drawn circles. Colonies selected with
Ampicillin.

![](http://parts.igem.org/wiki/images/2/2e/DH5ALPHA-EXO.jpg)

*Fig 3. Transformed DH5alpha with pCR™2.1-TOPO™ cloning vector +
BBa\_K4349005 construct. Colonies are shown inside the drawn circles. Colonies selected with
Ampicillin.*

**Digestion**

The bacteria grew and we performed Miniprep to obtain more quantity of the plasmid. After that, we
performed two digestions (in the Miniprep and in the pSB1C3): first with EcoRI and then with PstI. In the image below, we show a 1% agarose gel, where we confirmed the digestion with EcoRI and extracted the resultant bands from the gel. The stains in the gel are caused by RNA contamination, because we forgot to add RNAse during Miniprep.

![](http://parts.igem.org/wiki/images/0/08/Eletroforese-EcoRI-Endo.jpg)

*Fig 4. Agarose gel electrophoresis. Endochi (1241 bp) and exochi (1542 bp)
were digested with EcoRI and had the size as expected.*

Then, we digested the resultant samples with PstI. Then, we precipitated the post-digestion DNA samples.

**Ligation**

After that, we ligated the backbone + the insert with the T4 DNA Ligase. We left the reaction in the
Thermocycler 16°C overnight. In the subsequent day, we transformed BL21 *E. coli* .

![](http://parts.igem.org/wiki/images/b/bb/Endo-BL21-200ul.jpg)

*Fig 5. Transformed BL21 with pSB1C3 vector + BBa\_K4349007 construct.
Colonies are shown inside the drawn circles. Colonies selected with Chloramphenicol.*

![](http://parts.igem.org/wiki/images/d/d7/Exo-BL21-100ul.jpg)

*Fig 6. Transformed BL21 with pSB1C3 vector + BBa\_K4349005 construct.
Colonies are shown inside the drawn circles. Colonies selected with Chloramphenicol.*

**Confirmative PCR**

We also successfully performed PCR to confirm the insertion of our insert using Vf and Vr primers that
flank the gene regions.

![](https://static.igem.wiki/teams/4349/wiki/results/pcr.png)

*Fig 7. Agarose gel electrophoresis. PCR for confirmation of genes using VF2
and VR primers.*

**Expression**

In search of investigating the expression of our enzyme. We cultured 1 liter of each enzyme in LB, and
separated its supernatant and pellet. Afterward, we concentrated our supernatant, as our enzymes should be
secreted in theory. We then sonicated the pellet, separating the soluble and insoluble fractions to cover
all our possibilities. Then, we made a new polyacrylamide gel.

Unfortunately, it was not possible to add the pellet and insoluble fraction samples, because they did not
enter the gel, because of their viscosity. We can see that the solution fraction has demonstrated a drag
through the gel, probably caused by proteins native to E. coli. Sadly we didn't see the band corresponding
to our enzymes in any of the samples, even in the supernatant as expected.

![](https://static.igem.wiki/teams/4349/wiki/notebook/gel-05-10-22.png)

*Fig 8. Polyacrylamide gel.*

We assume that if our proteins are being secreted, there is a small amount, which cannot be visualized
on
the agarose gel. We assume that there is an expression of the proteins, in view of the altered visual
appearance of the transformation plating (Figs 5 and 6).
To confirm, we would have liked to have performed a Western Blotting test, but it was not possible due to
the short time.

## Expression of Endo and Exochitinase in *L. acidophilus*

Our main idea was to express and secrete both enzymes in *Lactobacillus acidophilus* and
characterize
signal peptides quantifying exported GFP. So we designed constructions totally optimized to produce and
send
the proteins to the extracellular compartment.

![](https://static.igem.wiki/teams/4349/wiki/results/circuito-endochi-lac-bba-k4349009.png)

![](https://static.igem.wiki/teams/4349/wiki/results/circuito-exochi-lac-bba-k4349008.png)

*Fig 9. Endochi (BBa\_K4349009) and exochi (BBa\_K4349008) construction optimized for  *L.
acidophilus.**

When our constructions arrived, we tried to clone them into pGEM®-T Easy vector, but it wasn't working
out
(Sep 5 to 9 - Week 1). Meanwhile, in our other lab space (UFMG), the group successfully cloned the
constructions optimized for  *E. coli*  into pCR™2.1-TOPO™ vector and then into pSB1C3. So they sent
us
a sample and we could transform the  *E. coli*  optimized constructions of Exo and Endochitinases in
*L. acidophilus*

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lac-endoufv.jpeg)

*Fig 10. Transformed L. acidophilus with pSB1C3 + BBa\_K4349009 construct.
Colonies selected with Chloramphenicol.*

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-lac-exo-ufv.jpeg)

*Fig 11. Transformed L. acidophilus with pSB1C3 + BBa\_K4349008 construct.
Colonies selected with Chloramphenicol.*

The transformed bacteria grew in 30ug/uL and in 50 ug/uL of Chloramphenicol

![](https://static.igem.wiki/teams/4349/wiki/results/lac-liq-clo.jpeg)

*Fig 12. Transformed L. acidophilus with pSB1C3 + BBa\_K4349009 or
BBa\_K4349008 constructs in liquid media with 50 ug/mL of Chloramphenicol.*

But when we did a plasmid extraction and a PCR, the samples showed highly unspecific bands, it was
probably an experimental mistake, but the deadline couldn’t allow us to repeat the experiment. However,
because
the bacteria is growing on MRS broth supplemented with 50 ug/mL of CLO, we believe that the chitinases
were
transformed.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-colony-pcr-lac-ufv.png)

*Fig 13. PCR of L. acidophilus with pSB1C3 + BBa\_K4349009 or BBa\_K4349008
constructs.*

Along with the PCR we were also trying to turn chitin, which is highly insoluble, in colloidal chitin to
add it to agar plates and check if *Lactobacillus* was secreting the enzymes. The colloidal chitin obtained
by
the protocol wasn’t as soluble as we expected but we could put it in a plate with the bacteria. The
results
suggested an initial halo formation that could be due to the degradation of chitin, but further tests need
to
be done.

![](https://static.igem.wiki/teams/4349/wiki/notebook/oct-halo-lac-ufv.png)

*Fig 14. Screening of chitinase production by L. acidophilus on
MRS-agar-chitin 2% plate.*

## Signal peptides for *L. acidophilus*

For the signal peptides, we assembled multiple constructions using the same promoter, RBS, GFP, and
terminator, with the variation of the signal peptide, the full collection can be found  [here](https://2022.igem.wiki/ufmg-ufv-brazil/part-collection). We also did a codon
optimization
of the signal peptide SacB form  *Bacillus subtilis*  to improve the secretion of extracellular
proteins.

But unfortunately, because of the failed cloning into pGEM®-T Easy, and the lack of TOPO in our lab, we
could not proceed with the signal peptides testing in enough time before the competition.

## Troubleshooting

Since our wet lab team faced many problems, we decided to make a troubleshooting session that can be
helpful for future iGEMer's that may come across similar situations

![](https://static.igem.wiki/teams/4349/wiki/results/results-troubleshooting.png)

# Docking

Our molecular docking simulations show that the chosen chitinases would bind correctly with a good
binding
affinity score to a chitin polymer (octamer).

*A. niger* CfcI bound to chitin shows a free energy score (ΔG(Kcal/mol)) of -11.6, pointing to a
favorable reaction with chitin binding to the protein pocket.

*P. chlamydosporia* Pchi44 docking results showed a free energy score of -10.6, also pointing to a
favorable reaction.

![](https://static.igem.wiki/teams/4349/wiki/protein-images/cfui-surface.png)

*Fig 15. CfcI chitinase bound to a chitin octamer.*

![](https://static.igem.wiki/teams/4349/wiki/protein-images/pchi44-surface.png)

*Fig 16. Pcchi44 chitinase bound to a chitin octamer.*

# Modelling

Through our mathematical model, we obtained different parameters from the literature and used them to
predict the chitinase production by *Lactobacillus acidophilus* inside the human intestine. To do
that, we created a function that represents the relation between probiotic factor and time. After
integrating this function and joining it with other parameters, we obtained the chitinase production by
our probiotic chassi inside the human intestine. Dividing the resultant value by the intestinal volume, we
achieved the concentration of the chitinase inside the small intestine, according to time and to the
probiotic dose. We performed this calculation both with endochitinase and exochitinase, and using the
maximum and minimum values of our variables, obtaining our results as maximum and minimum concentration as
well.

##### Additional information can be found [HERE.](https://2022.igem.wiki/ufmg-ufv-brazil/model)

# Hardware

The development was an open-source modular low-cost benchtop bioreactor. It stands autoclave (except for
pH sensor which is chemically sterilized using a hypochlorite solution solution). It is able to read pH,
temperature, and proxy Optical Density through the reading of the media color changes. Users can also control
light exposure
(it can be used as a photo-bioreactor) and activate peristaltic pumps to compensate for pH changes or to keep the
growth phase steady. A heating pad is also an option to keep it warm, if not using it as a
photo-bioreactor.

##### Additional information can be found [HERE.](https://2022.igem.wiki/ufmg-ufv-brazil/hardware)