Notebook

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

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

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

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

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.

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.

Participants:Livia Galinari, Luiza Possa, Arthur Enrici

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.

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.

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.

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:

One of the BDA plates was contaminated.

Participants: Vanessa Pasa, Yala Sampaio, Paulo Muniz

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:

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.

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!

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.

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.

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.

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:

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:

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

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.

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.

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!

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:

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:

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:

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:

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!!!

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.

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:

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.

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.

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%.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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