iGEM 2021 — Team UNILA_LatAm

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# Team:UNILA LatAm/Notebook

Notebook | iGEM UNILA\_LatAm

[![](https://static.igem.org/mediawiki/2021/9/98/T--UNILA_LatAm--Logo.png)](https://2021.igem.org/Team%3AUNILA_LatAm)

* Project

  [Description](https://2021.igem.org/Team%3AUNILA_LatAm/Description)[Design Overview](https://2021.igem.org/Team%3AUNILA_LatAm/Design_Overview)[Background](https://2021.igem.org/Team%3AUNILA_LatAm/Background)[Engineering](https://2021.igem.org/Team%3AUNILA_LatAm/Engineering)[Contribution](https://2021.igem.org/Team%3AUNILA_LatAm/Contribution)
* DryLab

  [Design](https://2021.igem.org/Team%3AUNILA_LatAm/Design)[Model](https://2021.igem.org/Team%3AUNILA_LatAm/Model)
* WetLab

  [Experiments](https://2021.igem.org/Team%3AUNILA_LatAm/Experiments)[Results](https://2021.igem.org/Team%3AUNILA_LatAm/Results)[Notebook](https://2021.igem.org/Team%3AUNILA_LatAm/Notebook)[Protocols](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols)[Parts](https://2021.igem.org/Team%3AUNILA_LatAm/Parts)[Safety](https://2021.igem.org/Team%3AUNILA_LatAm/Safety)
* Human Practices

  [Introduction](https://2021.igem.org/Team%3AUNILA_LatAm/Human_Practices)[Sustainability](https://2021.igem.org/Team%3AUNILA_LatAm/Sustainability)[Implementation](https://2021.igem.org/Team%3AUNILA_LatAm/Implementation)[Fighting Underreporting](https://2021.igem.org/Team%3AUNILA_LatAm/Fighting_Underreporting)[Integrated Human Practices](https://2021.igem.org/Team%3AUNILA_LatAm/Integrated_Human_Practices)[Education](https://2021.igem.org/Team%3AUNILA_LatAm/Education)[Inclusivity](https://2021.igem.org/Team%3AUNILA_LatAm/Inclusivity)[Communication](https://2021.igem.org/Team%3AUNILA_LatAm/Communication)[Funding](https://2021.igem.org/Team%3AUNILA_LatAm/Funding)
* Team

  [Team Members](https://2021.igem.org/Team%3AUNILA_LatAm/Team)[Attributions](https://2021.igem.org/Team%3AUNILA_LatAm/Attributions)[Collaborations](https://2021.igem.org/Team%3AUNILA_LatAm/Collaborations)[Partnership](https://2021.igem.org/Team%3AUNILA_LatAm/Partnership)[Sponsors](https://2021.igem.org/Team%3AUNILA_LatAm/Sponsors)
* Awards

  [Medal Criteria](https://2021.igem.org/Team%3AUNILA_LatAm/Medal_Criteria)

# Notebook

---

##### Contents

# Week 1 (02/15/2021 - 02/19/2021)

## Day: 02/15/2021

Experiment: Bacillus subtilis 168 reactivation. Objectives: Reactivation of Bacillus subtilis 168. Responsible: Maria Barmaimon and Giulio Braatz Description: We received plates of Bacillus subtilis 168 from Danielli Pedrolli (UNESP), and the isolated colonies were reactivated.

## Day: 02/16/2021

Experiment: Bacillus subtilis 168 cultivation and preservation. Objectives: Reactivation of Bacillus subtilis 168 stocks. Responsible: Maria Barmaimon Description: Continuation of the day before, the colonies didn't grow.

## Day: 02/18/2021

Experiment: Reactivation of stocks. Objectives: Reactivation of Bacillus subtilis 168 stocks. Responsible: Matheus Araujo and Giulio Braatz Description: Cultivation from stocks.

# Week 2 (02/22/2021 - 02/26/2021)

## Day: 02/23/2021

Experiment: Preservation of Bacillus subtilis 168. Objectives: Preservation in glycerol stocks. Responsible: Maria Barmaimon. Description: Stocks made from the cultivation initiated on 02/18.

# Week 3 (03/22/2021 - 03/26/2021)

## Day: 03/22/2021

Experiment: Medium preparation and bacterium reactivation. Objectives: Reactivation from stabs. Responsible: Matheus Araujo and Gabriela Milena. Description: Preparation of media with antibiotic on liquid media the strains containing plasmids: **DH10beta-pJN105, DH10beta-pSEVA231, DH10beta-pBBR1MCS-2, DH10beta-pSB1K3** sent by Eric Velasco from USP. Protocols: P001A and P004A.

## Day: 03/23/2021

Experiment: Bacterium reactivation. Objectives: Reactivation from liquid media to solid media on plates of the strains containing plasmids. Responsible: Matheus Araujo and Gabriela Milena. Description: Continuation of the last day. Protocols: P004A

## Day: 03/24/2021

Experiment: Bacterium reactivation. Objectives: Cultivation from plates to liquid media of DH10beta-pJN105, DH10beta-pSEVA231, DH10beta-pBBR1MCS-2, DH10beta-pSB1K3. Responsible: Matheus Araujo and Gabriela Milena. Description: The inoculum was done from isolated colonies. Protocols: P004A

## Day: 03/26/2021

Experiment: Bacterium preservation. Objectives: Preservation of reactivated bacterias E. coli DH10beta-pJN105, DH10beta-pSEVA231, DH10beta-pBBR1MCS-2, DH10beta-pSB1K3. Responsible: Maria Barmaimon. Description: The bacterium reactivated were preserved. Protocols: P003A

# Week 4 (04/12/2021 - 04/16/2021)

## Day: 04/13/2021

Experiment: Solutions preparation. Objectives: Preparation of stock solutions for use in multiple activities. Responsible: Matheus Araujo and Giulio Braatz. Description: Stock solutions were prepared. Protocols: P005A.

# Week 5 (06/07/2021 - 06/11/2021)

## Days: 06/08/2021 and 06/09/2021

Experiment: Preparation of miniprep solutions. Objectives: Preparation of solutions that we'll be used for miniprep. Responsible: Matheus Araujo Description: Stock solutions were prepared. Protocols: P005A and P005B.

# Week 6 (06/14/2021 - 06/18/2021)

## Days: 06/14/2021 to 06/16/2021

Experiment: Medium preparation and reactivation of Escherichia coli DH10B with pBBR1MCS-2 plasmid. Objectives: Reactivation from stock. Responsible: Matheus Araujo and Gabriela Milena. Description: Medium preparation and bacterium reactivation. Protocols: P001A and P004A.

## Day: 06/17/2021

Experiment: Alkaline lysis miniprep. Objectives: Extraction of pBBR1MCS-2 plasmid from E. coli DH10B. Responsible: Matheus Araujo Description: Miniprep followed protocol and was quantified using nanodrop. Protocols: P006A.

## Day: 06/18/2021

Experiment: Agarose gel electrophoresis. Objectives: Electrophoresis with miniprep product. Responsible: Matheus Araujo Description: The electrophoresis run followed the protocol. Protocols: P009A.

# Week 7 (08/30/2021 - 09/03/2021)

## Day: 08/30/2021

Experiment: Preparation LB medium for selection plates and materials for competence test. Objectives: Preparation of solid LB with ampicillin and sterilizing of the necessary materials Responsible: Matheus Araujo. Description: The plates, microtubes, tips, and LB medium were autoclaved. The antibiotic was added to the medium after it got cold on the laminar flow. Protocols: P001A.

## Day: 09/01/2021

Experiment: Competence test of E. coli DH5-alpha and obtention of plasmid pUC19 in bacteria Objectives: Test the transformation efficiency of the commercial bacteria, E. coli DH5-alpha, and transform with empty backbone pUC19. Responsible: Matheus Araujo. Description: The transformation occurred according to the protocol. After recovery, the plating was done with 100microL to duplicate dilution. We used 10 microL of plasmid, different from what the protocol indicated because of a stock error. Protocols: P002A.

## Day: 09/02/2021

Experiment: Transformation efficiency calculation and cultivation of isolated colonies. Objectives: To calculate the efficiency of the transformation executed on the day before and initiate the cultivation to preserve the empty backbone strain. Responsible: Matheus Araujo. Description: After 16-20 hours, we counted the colonies from the 10^-1 dilution and calculated the TE to obtained 5 x 10^8 cfu/µg. Protocols: P004A.

## Day: 09/03/2021

Experiment: E. coli DH5-alpha with pUC19 preservation Objectives: Preserve E. coli DH5-alpha with the empty backbone pUC19. Responsible: Matheus Araujo. Description: The preservation occurred according to the protocol. Protocols: P003A.

# Week 8 (09/13/2021 - 09/17/2021)

## Day: 09/13/2021

Experiment: Reactivation of Escherichia coli BL21 (D3). Objectives: To initiate the reactivation of E. coli BL21 (D3). Responsible: Matheus Araujo. Description: The reactivation followed the protocol. The cultures grew for 4 hours, and 100 ml of the cultivation was transferred and distributed to the plates. Protocols: P004A.

## Days: 09/14/2021 to 09/16/2021

Experiment: Preparation of the inducted chemo competency of Escherichia coli BL21 (D3). Objectives: Preparation of solutions, cellular cultivation, and other necessary materials for the induction. Responsible: Matheus Araujo. Description: The stocks were contaminated, so it took a little longer than we anticipated. We isolated our strain and continued with procedures. Protocols: P004A.

## Day: 09/17/2021

Experiment: Preserve and Induce chemo competency of Escherichia coli BL21 (D3). Objectives: Use overnight cultivation to make new glycerol stocks and induce chemo competency in E. coli BL21 (D3). Responsible: Matheus Araujo. Description: The preservation and induction were done according to protocols. Protocols: P003A and P007A.

# Week 9 (09/20/2021 - 09/24/2021)

## Day: 09/21/2021

Experiment: Preparation of solid LB medium with kanamycin. Objectives: Prepare and sterilize the medium. Responsible: Matheus Araujo. Description: The medium was prepared according to protocol. Protocols: P001A

## Day: 09/23/2021

Experiment: Competency test of Escherichia coli DH10B e BL21 (D3). Objectives: Test the transformation efficiency of Escherichia coli DH10B e BL21 (D3). Responsible: Matheus Araujo. Description: We made a serial dilution with the pBBR1MCS-2 plasmid miniprep 2, using its total volume in 100 microl of TE, and the following concentrations were measured, \*1: 495 ng/microl; 2: 147 ng/microl; \*3: 35 ng/microl; \*14 ng/microl.The transformation process followed the protocol. We used the second dilution of pBBR1MCS-2 to transform BL21 (D3) and Dh10B and the plasmid pUC19 to transform BL21(D3). Protocols: P002A.

## Day: 09/24/2021

Experiment: Counting of CFU to calculate transformation efficiency. Objectives: Count CFU and calculate the efficiency transformation of Escherichia coli BL21 (D3). Responsible: Matheus Araujo. Description: The only successful transformation was with the plasmid pUC19 in E. coli BL21 (D3). We counted the colonies of two dilutions, 10^-1 e 10^-2, and obtained a TE of 1,32 x 10^7 cfu/microg. 1,4 x 10^7 cfu/microg.

# Week 10 (09/27/2021 - 10/02/2021)

## Day: 09/27/2021

Experiment: Reactivation and materials preparation. Objectives: Reactivate Escherichia coli DH10B for miniprep of the pBBR1MCS-2 plasmid. Responsible: Matheus Araujo. Description: Reactivation started through striation from the stock. Protocols: P004A.

## Day: 09/28/2021

Experiment: Reactivation and miniprep. Objectives: To cultivate Escherichia coli DH10B, do a miniprep of the pBBR1MCS-2 plasmid and quantify it. Responsible: Matheus Araujo. Description: The miniprep was done from 3 ml of culture in two microtubes according to protocol, using 35 ul for elution. We measured the miniprep yield by quantifying plasmid in the microdrop, getting the following concentrations. 1: 26 ng/microl (A260/A280: 2.11; A260/A230: 3.36) 2: 30 ng/microl (A260/A280: 1.96; A260/A230: 2.09) Protocols: P004A and P006B

## Day: 09/30/2021

Experiment: Resuspension of primers, PCR for linearization, and electrophoresis. Objectives: Resuspend primers and linearize the pBBR1MCS-2 plasmid through PCR and visualize it by electrophoresis. Responsible: Matheus Araujo. Description: We dilute 10 µL of 1 M tris in 990 microL of nuclease-free water. The solution was used to suspend the primers pBBR1MCS-2/pUC19 - REV and pBBR1MCS-2/pUC19 - FWD, at the stock concentration of 100 ng/microL, 26 microL was added in pBBR1MCS-2/pUC19 - FWD containing 26.1 nmol and 30 microL in pBBR1MCS-2/pUC19 - REV containing 29.7 nmol. The next step was the preparation of the working solution at a concentration of 0.5 microg/microL, where 1 microL of the stock primers was added in 7 microL of the 10 mmol tris solution. We made a PCR reaction and electrophoresis. The samples used were: products from the miniprep of 09/29 (pBBBR1MCS-2: 1:26 ng/microL and 2: 30 ng/microL) 3 microL each, PCR sample (+) 2 microL and (-) 3 microL, miniprep product from alkaline lysis (pBBR1MCS-2: 1:1700 ng/microL and 2: diluted to 1:495 ng/microl) with 2 and 3 microl, respectively, and 5 microl of DNA ladder. Protocols: P008A and P009A.

## Day: 10/01/2021

Experiment: Electrophoresis and Purification. Objectives: Run electrophoresis with PCR product and purify it through gel extraction. Responsible: Matheus Araujo. Description: Electrophoresis occurred according to protocol, and one of the DNA fragments was purified. The quantification showed inconclusive DNA presence. Protocols: P009A and P010A.

## Day: 10/02/2021

Experiment: PCR for linearization and electrophoresis Objectives: Linearize the pBBR1MCS-2 plasmid through PCR and visualize it by electrophoresis. Responsible: Matheus Araujo. Description: The preparation and execution of the PCR occurred according to protocol. The volumes used were 1 microL for primers and template (total 30 ng). Two templates were used (a dilution of aliquot 1 of the first miniprep ~30ng/microL microdrop). The DNA stain was added later and kept in incubation until 10/04 to visualize the DNA fragments. Protocols: P008A and P009A.

# Week 11 (10/04/2021 - 10/08/2021)

## Day: 10/04/2021

Experiment: Reactivation, Electrophoresis, and Purification. Objectives: Reactivate the bacteria with the pBBR1MCS-2 plasmid, run electrophoresis with PCR product from day 10/02, and purify it through gel extraction. Responsible: Matheus Araujo. Description: The reactivation started with an hour 1 ml cultivation then transferred to the plate. We made electrophoresis and extraction of DNA fragments of the gel. The quantification showed inconclusive DNA presence. Protocols: P004A, P009A and P010A.

## Day: 10/05/2021

Experiment: Reactivation, Electrophoresis, and PCR. Objectives: Reactivate the bacteria with the pBBR1MCS-2 plasmid, execute PCR with purified fragments and electrophoresis. Responsible: Matheus Araujo. Description: We kept with the reactivation, later executed a PCR reaction using 1 microL of the plasmid obtained in the first miniprep and finalized with electrophoresis. Protocols: P004A

## Day: 10/07/2021

Experiment: Molecular Marker's preparation and electrophoresis. Objectives: Prepare molecular weight markers through enzyme digestion and execute electrophoresis. Responsible: Matheus Araujo. Description: We digest Lambda DNA using EcoR I and the plasmid pBBR1MCS-2 with Bbs I, both were incubated for two hours at 37 graus C and inactivated at 65 graus C for 15 min. We changed the electrophoresis buffer using TAE 1X, and DNA labeling happened after the run by incubation. Protocols: P009A

Experiment: Bacteria reactivation. Objectives: Reactivate Escherichia. coli BL21 (pLyss) for preservation on glycerol stock. Responsible: Gabriela Challco Description: The preservation of Escherichia. coli BL21 (pLyss) sent by Eric Velasco from USP started in the morning, and the incubation on plates in the evening. Protocols: P004A

## Day: 10/08/2021

Experiment: Electrophoresis, PCR, and gel purification. Objectives: Analysis of the electrophoresis run, PCR, and purification of the gel fragments. Responsible: Matheus Araujo. Description: The analysis wasn't possible for DNA labeling failure, requiring a new electrophoresis gel. The new electrophoresis still wasn't satisfactory. So we did a new PCR reaction. Protocols: P009A, P008A and P010A.

Experiment: Bacteria preservation. Objectives: Reactivate and preserve E. coli BL21 (pLyss) on glycerol stock. Responsible: Gabriela Challco Description: The cultivation in liquid media started in the morning and the preservation of E. coli BL21 (pLyss) in the evening. Protocols: P003A

# Week 12 (10/09/2021 - 10/16/2021)

## Day: 10/09/2021

Experiment: PCR amplification, assembly, and transformation. Objectives: Amplify the plasmid purified from the gel by PCR in the assembly reaction and transform it into E. coli DH5-alpha and BL21. Responsible: Matheus Araujo Description: The purified plasmids from the gel were measured in microdrop: 22 ng/microL and 11 ng/microL. We used 1 microL of each to make a PCR reaction. The gBlocks G1 and G0 were resuspended in TE to a concentration of 10 ng/microL. The PCR product and the 22 ng/microL linear plasmids was used for the assembly reaction. We carried out eight transformations, 2 for each assembly, using E. coli DH5-alpha and BL21, and the entire volume of the recovery culture was plated on three selection plates for each transformation. Protocols: P001A, P008A, P009A, P011A and P002A.

## Day: 10/10/2021

Experiment: Cultivation of selected colonies. Objectives: Identify isolated colonies from selection plates after assembly, and cultivate them in a liquid medium. Responsible: Matheus Araujo Description: All the assemblies except one presented colony grow. Cultivations were done with isolated colonies. The cultivation was incubated for 16 hours at 28 graus C 130 RPM. Protocols: P004A

## Day: 10/11/2021

Experiment: PCR, miniprep and colony PCR. Objectives: Miniprep of cultivation with E. coli DH5-alpha PCR reaction and confirmation by electrophoresis. Responsible: Matheus Araujo Description: First, we resuspended the primers for inserts amplification to 100 ng/microL and 0.5 microg/microL, then made miniprep with three cultivations containing the G1 and G0 construct. We made a PCR reaction (annealing temperature to 65 graus C, the extension time was 35s and 2 min of final extension) with the miniprep products and used as a positive control the stock of G0 construct. Three isolated DH5-alfa colonies were used to start cultivation in a liquid media and a colony PCR. Electrophoresis using the PCR products was done to confirm the assembly. With the miniprep products, we did a recloning of G1 construct in E. coli BL21. All the recovery content was plated. Protocols: P006B, P008A, P009A and P002A.

## Day: 10/12/2021

Experiment: PCR, miniprep and IPTG preparation. Objectives: Miniprep of the cultivation with G0 E. coli DH5-alpha and confirmation PCR. Preparation of 1M IPTG aliquots. Responsible: Matheus Araujo Description: We did miniprep from the cultivation of DH5-alfa (with G0) following protocol and then a PCR with the miniprep products. A colony PCR was also done to confirm the recloning of G1 in BL21, followed by an electrophoresis run. After PCR confirmation, we recloned G0 assembly in BL21. Protocols: P006B, P008A, P009A and P002A.

Experiment: Preparation of LB media and bacteria reactivation. Objectives: Prepare liquid and solid LB media to isolate G1 E. coli DH5-alpha, confirm the preservation of E.coli BL21 (plyss), reactivate E.coli DH10B containing the pBS0e plasmid. Responsible: Gabriela Challco Description: Medium preparation with antibiotics, kanamycin, and chloramphenicol. The first step towards reactivation was bacterium cultivation. E.coli DH10B pBSθe was cultivated from an isolated colony in a falcon tube; plyss BL21 was inoculated on plates from stocks, and G1 E. coli DH5-alpha was grown in solid media. Protocols: P001A and P004A

## Day: 10/13/2021

Experiment: Bacteria reactivation. Objectives: Reactivation of G1 E. coli DH5-alpha and E. coli DH10B containing pBS0e plasmid. Responsible: Gabriela Challco Description: The E. coli DH10B was inoculated on plates and G1 E. coli DH5-alpha transferred to liquid media. The growth of E. coli BL21 (plyss) colonies confirmed the availability of the stock.

Experiment: Solutions preparation and expression cultivation. Objectives: Preparation of 1M IPTG stocks and TSE solution. Begin of expression cultivation. Responsible: Matheus Araujo Description: The IPTG stock was done from 1,19g of lyophilized IPTG and 5ml of nuclease-free water, filtered and stored at -80 graus C. A 1M Sucrose solution also was done, followed by the TSE solution. The expression cultivation began at 02 pm following protocol. The induction occurred at 07:20 pm and when the OD was at 0.72 the rotation was altered to 200 RPM. Protocols: P005A and P012A.

## Day: 10/14/2021

Experiment: AMP production and trypsin treatment. Objectives: AMP production and activation trypsin treatment. Responsible: Matheus Araujo and Michelli Teixeira. Description: The expression cultivation was transferred to 12 microtubes (because our centrifuge for polypropylene tubes didn't reach the needed RPM), then the AMP obtention experiment followed the protocol. Later we started the trypsin treatment as described in the protocol. We used PBS (1X) to complete the periplasmatic product to 22 ml of volume equal to the supernatant. Both treatments and trypsin were filtered in a 0,22 microm pore. Protocols: P013A and P014A

Experiment: Reactivation and preservation. Objectives: Reactivation and preservation of strains. Responsible: Gabriela Challco. Description: G0 E. coli DH5-alpha and E. coli DH10B-pBSθe were transferred to liquid media. The reactivation of DH5 alfa pBS1C and G1 E. coli BL21 moved on. The G1 E. coli DH5-alpha was preserved. Protocols: P003A and P004A

## Day: 10/15/2021

Experiment: Reactivation, preservation, and Miniprep. Objectives: Reactivation of E.coli DH10B pBSθe and G1 BL21. Preservation of G0 DH5 alfa and E.coli DH10B pBSθe. Miniprep of G0 DH5-alfa. Responsible: Matheus Araujo and Gabriela Challco. Description: E.coli DH10B pBSθe and G0 E. coli DH5-micro were preserved with 500 microL of glycerol and 500 microL of cultivation medium. The reactivation of the E.coli E.coli DH10B pBSθe and the G1 E.coli BL 21 followed protocol. The miniprep was done from 3 mL of the G0 E. coli DH5-micro cultivation. Protocols: P003A, P004A and P006B.

Experiment: Leishmanicidal assay. Objectives: Counting leishmania cells, and transfer 1x10^5 to tubes with media, and start the treatment. Responsible: Matheus Araujo and Michelli Teixeira. Description: We counted leishmania cells, transferred them to tubes with media, and added the treatments before inoculating.

## Day: 10/16/2021

Experiment: Preservation and recloning of BL21 with G0 assembly. Objectives: Preservation of E.coli DH10B pBSθe and G1 BL21. Cloning of BL21 with G1. Responsible: Matheus Araujo and Gabriela Challco. Description: The preservation of E.coli DH10B pBSθe and G1 BL21 was made with glycerol 50%, following protocol. The recloning used 10 microL of plasmid from the previous miniprep with an approximate yield of 21 and 14 ng/microL. 400 microL of volume was distributed in 4 selection plates, following protocol. Protocols: P003A and P002A

Experiment: Leishmania counting cells. Objectives: Counting of Leishmania cells in every assay cultivation. Responsible: Matheus Araujo e Michelli Teixeira. Description: The cultivation did not grow as expected, making it difficult to identify viable cells. The activity was postponed to the next day. Protocols: P015A.

## Day: 10/17/2021

Experiment: Leishmania counting cells and update on recloned G0 E. coli BL21. Objectives: Counting of Leishmania cells in every assay cultivation and see the status on selection plates. Responsible: Matheus Araujo. Description: The G0 E. coli BL21 cells didn't grow, and the plates were incubated for another 24 hours. The cultivation did not grow as expected, making it difficult to identify viable cells. The activity was postponed to the fifth day of cultivation. Protocols: P015A.

## Day: 10/20/2021

Experiment: Leishmania counting cells. Objectives: Counting of Leishmania cells in every assay cultivation. Responsible: Matheus Araujo. Description: We attempted to count the cells and concluded that there was no significant growth. The medium used wasn't in the ideal conditions, and the cultivation presented contamination. The experiment has been canceled. Protocols: P015A.

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# Team:UNILA LatAm/Engineering

Engineering | iGEM UNILA\_LatAm

[![](https://static.igem.org/mediawiki/2021/9/98/T--UNILA_LatAm--Logo.png)](https://2021.igem.org/Team%3AUNILA_LatAm)

* Project

  [Description](https://2021.igem.org/Team%3AUNILA_LatAm/Description)[Design Overview](https://2021.igem.org/Team%3AUNILA_LatAm/Design_Overview)[Background](https://2021.igem.org/Team%3AUNILA_LatAm/Background)[Engineering](https://2021.igem.org/Team%3AUNILA_LatAm/Engineering)[Contribution](https://2021.igem.org/Team%3AUNILA_LatAm/Contribution)
* DryLab

  [Design](https://2021.igem.org/Team%3AUNILA_LatAm/Design)[Model](https://2021.igem.org/Team%3AUNILA_LatAm/Model)
* WetLab

  [Experiments](https://2021.igem.org/Team%3AUNILA_LatAm/Experiments)[Results](https://2021.igem.org/Team%3AUNILA_LatAm/Results)[Notebook](https://2021.igem.org/Team%3AUNILA_LatAm/Notebook)[Protocols](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols)[Parts](https://2021.igem.org/Team%3AUNILA_LatAm/Parts)[Safety](https://2021.igem.org/Team%3AUNILA_LatAm/Safety)
* Human Practices

  [Introduction](https://2021.igem.org/Team%3AUNILA_LatAm/Human_Practices)[Sustainability](https://2021.igem.org/Team%3AUNILA_LatAm/Sustainability)[Implementation](https://2021.igem.org/Team%3AUNILA_LatAm/Implementation)[Fighting Underreporting](https://2021.igem.org/Team%3AUNILA_LatAm/Fighting_Underreporting)[Integrated Human Practices](https://2021.igem.org/Team%3AUNILA_LatAm/Integrated_Human_Practices)[Education](https://2021.igem.org/Team%3AUNILA_LatAm/Education)[Inclusivity](https://2021.igem.org/Team%3AUNILA_LatAm/Inclusivity)[Communication](https://2021.igem.org/Team%3AUNILA_LatAm/Communication)[Funding](https://2021.igem.org/Team%3AUNILA_LatAm/Funding)
* Team

  [Team Members](https://2021.igem.org/Team%3AUNILA_LatAm/Team)[Attributions](https://2021.igem.org/Team%3AUNILA_LatAm/Attributions)[Collaborations](https://2021.igem.org/Team%3AUNILA_LatAm/Collaborations)[Partnership](https://2021.igem.org/Team%3AUNILA_LatAm/Partnership)[Sponsors](https://2021.igem.org/Team%3AUNILA_LatAm/Sponsors)
* Awards

  [Medal Criteria](https://2021.igem.org/Team%3AUNILA_LatAm/Medal_Criteria)

# Engineering

---

##### Contents

# Leishmaniasis and Synthetic Biology

Before we started our circuit's design, we searched for efficient strategies to treat parasitic diseases. These strategies are a big challenge to leishmaniasis, considering that the generalist and diversified way of the parasite's infection demands robust treatment options. We were introduced to paratransgenesis in a meeting with specialists and advisors, learning it was a way to control the parasite's transmissibility with engineered bacteria into the vector population. This technique was explored by iGEM USP 2017 and motivated us to design our paratransgenic platform to fight leishmaniasis.

BioPank's engineering cycle expanded to different modules, each one of them designed to make the traditional paratransgenesis technique more robust, being ready to face the adversities of the real world. Considering all the problems that could arise in a real-world application, we traced strategies that led us to design four different genetic circuit modules and utilized different in silico and in vitro approaches to assess each one of them.

# Modules Design

![](https://static.igem.org/mediawiki/2021/a/ae/T--UNILA_LatAM--modules_engineering.png)

The circuits modularization favored a robust technique that would demand planning of "design, Model, Build and Test" for each module. This design framework allowed us to initiate four different engineering cycles to Protection, Detection, Overview, and Modeling. However, we completed the engineering cycle only for the Elimination Module because of our difficulty going to the lab due to the COVID-19 pandemic.

# Engineering Success and Elimination Module

The Elimination Module is responsible for acting against *Leishmania* inside the vector's midgut. Its primary function is to produce an effector molecule, the antimicrobial peptide (AMP) DRS-N1, in its deactivated form (proAMP), linked to an acidic pro-peptide, which will be separated at the moment of enzymatic digestion in the midgut. This mechanism was assessed in the lab by the expression of proAMP, trypsinization assay, and bioassay against Leishmania.

## DESIGN

The in vitro production of recombinant antimicrobial peptides in bacteria is a challenge. In a specific concentration, these peptides can considerably harm the chassis growth and derail its production [1]. Now imagine an engineered bacteria in the sandfly's midgut to produce enough AMPs to kill any parasitic cell. Probably, high quantities would diminish the vector's persistency and affect the bacteria's efficiency action. This context could put at risk the paratransgenesis technique, and that's why we have a strategy to avoid the situation.

In a talk with specialists, we realized that it was possible to produce these AMPs in a deactivated form, connected to pro-peptides that neutralize the cationic charge [2], and to induce their activation through enzymatic digestion that will split them up. We chose trypsin, an already highly present enzyme in the *Luztomyia longipalpis* midgut that rises its production after the blood meal.

The final Elimination Module design has the leishmanicidal activity AMPs and its related pro-peptides, according to the table:

| Pro-peptide | AMP | Final Composite |
| --- | --- | --- |
| [DRS-H3 pro-peptide](http://parts.igem.org/Part%3ABBa_K4075001) | [DRS-N1](http://parts.igem.org/Part%3ABBa_K4075004) | [BBa\_K4075008](http://parts.igem.org/Part%3ABBa_K4075008) |
| [E10 pro-peptide](http://parts.igem.org/Part%3ABBa_K4075002) | [CAM-W](http://parts.igem.org/Part%3ABBa_K4075005) | [BBa\_K4075009](http://parts.igem.org/Part%3ABBa_K4075009) |
| [DRS-S1 pro-peptide](http://parts.igem.org/Part%3ABBa_K4075003) | [DRS-S1](http://parts.igem.org/Part%3ABBa_K4075006) | [BBa\_K4075010](http://parts.igem.org/Part%3ABBa_K4075010) |
| [DRS-H3 pro-peptide](http://parts.igem.org/Part%3ABBa_K4075001) | [DRS-H3](http://parts.igem.org/Part%3ABBa_K4075007) | [BBa\_K4075011](http://parts.igem.org/Part%3ABBa_K4075011) |
| [E10 pro-peptide](http://parts.igem.org/Part%3ABBa_K4075002) | [DRS-N1](http://parts.igem.org/Part%3ABBa_K4075004) | [BBa\_K4075012](http://parts.igem.org/Part%3ABBa_K4075012) |

The circuit control is done by the Detection Module and for the implementation in *Bacillus subtilis*.

Nonetheless, the leishmanicidal AMPs have trypsin cleavage sites, harming any AMP strategy. Our team discussed this aspect with specialists who pointed out that the pro-peptides provide protection against digestion and raise the stability of the AMP. This corroborates with the idea of activating our system through limited ingestion, a natural event in the production of AMPs in different organisms [3].

Therefore, we decided to assess the proAMP activation through trypsinization and its return to a leishmanicidal activity through a cytotoxic assay.

## MODEL

From our circuit's design, we assessed the paratransgenesis system operation through a kinetic model. We saw that one of the main factors that could influence our system efficiency is the AMP cooperativity. This factor showed us the importance of testing the elimination module to assess the kinetic action characteristics of AMPs.

## TEST

Despite our original chassis being *Bacillus subtilis*, we decided to express the proAMPs in *E. coli* BL21(DE3) due to available time and lab resources. This fact did not disturb our goals since the strategy was to assess the AMP activation and deactivation. The selected AMPs had pro-peptides inducing their neutralization and the OmpA [4] signal peptide, with the function of secreting to the extracellular region or allocating the peptide in the periplasm. We used natural pro-peptides for the dermaseptins (DRSs) and a synthetic pro-peptide model for the CAM-W. As the promoter, we chose PT7\_LacO (BBa K2406020) that activates with IPTG presence.

The cloning of our circuit containing the proDRS-N1 (BBa\_K4075008) and the circuit with the reporter gene was made in NEB® 5-alpha Competent E. coli. In E. coli BL21(DE3), we succeeded only in the cloning of the proDRS-N1 construct.

With the assembly and cloning done, we obtained the proAMP, leading us to the final steps of our experiments. Those consisted of the enzymatic treatment with trypsin and leishmanicidal assay with the previous treatment product. We had three goals to demonstrate: 1) our effector molecule activation capacity in the presence of trypsin; 2) the relation between trypsin concentration and activation through partial digestion; and 3) the activated AMP leishmanicidal activity.

# References

1. JI, S. et al. Efficient biosynthesis of a Cecropin A-melittin mutant in *Bacillus subtilis* WB700. Nature Scientific Reports, 7(40587), 2017. <https://www.nature.com/articles/srep40587>
2. JUNIOR, N. G. O. et al. An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of crotalicidin fragment. Nature Scientific Reports, 8(11127), 2018. <https://www.nature.com/articles/s41598-018-29444-0>
3. Mahlapuu, M. et al. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Frontiers in Cellular and Infection Microbiology. 6:194, 2016. <https://www.frontiersin.org/articles/10.3389/fcimb.2016.00194/full>
4. Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of *B. subtilis* chitosanase enzyme from E. coli expression system. SpringerPlus, 5:1200, 2016. <https://springerplus.springeropen.com/articles/10.1186/s40064-016-2893-y>

A Project by

![](https://static.igem.org/mediawiki/2021/d/dc/T--UNILA_LatAM--LogoV3.png)

Contact

synfronteras@gmail.com

Sponsor

[![](https://static.igem.org/mediawiki/2021/8/89/T--UNILA_LatAM--LogoIPH.png)](https://www.itaipu.gov.br/)

Follow us!

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# Team:UNILA LatAm/Experiments

Experiments | iGEM UNILA\_LatAm

[![](https://static.igem.org/mediawiki/2021/9/98/T--UNILA_LatAm--Logo.png)](https://2021.igem.org/Team%3AUNILA_LatAm)

* Project

  [Description](https://2021.igem.org/Team%3AUNILA_LatAm/Description)[Design Overview](https://2021.igem.org/Team%3AUNILA_LatAm/Design_Overview)[Background](https://2021.igem.org/Team%3AUNILA_LatAm/Background)[Engineering](https://2021.igem.org/Team%3AUNILA_LatAm/Engineering)[Contribution](https://2021.igem.org/Team%3AUNILA_LatAm/Contribution)
* DryLab

  [Design](https://2021.igem.org/Team%3AUNILA_LatAm/Design)[Model](https://2021.igem.org/Team%3AUNILA_LatAm/Model)
* WetLab

  [Experiments](https://2021.igem.org/Team%3AUNILA_LatAm/Experiments)[Results](https://2021.igem.org/Team%3AUNILA_LatAm/Results)[Notebook](https://2021.igem.org/Team%3AUNILA_LatAm/Notebook)[Protocols](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols)[Parts](https://2021.igem.org/Team%3AUNILA_LatAm/Parts)[Safety](https://2021.igem.org/Team%3AUNILA_LatAm/Safety)
* Human Practices

  [Introduction](https://2021.igem.org/Team%3AUNILA_LatAm/Human_Practices)[Sustainability](https://2021.igem.org/Team%3AUNILA_LatAm/Sustainability)[Implementation](https://2021.igem.org/Team%3AUNILA_LatAm/Implementation)[Fighting Underreporting](https://2021.igem.org/Team%3AUNILA_LatAm/Fighting_Underreporting)[Integrated Human Practices](https://2021.igem.org/Team%3AUNILA_LatAm/Integrated_Human_Practices)[Education](https://2021.igem.org/Team%3AUNILA_LatAm/Education)[Inclusivity](https://2021.igem.org/Team%3AUNILA_LatAm/Inclusivity)[Communication](https://2021.igem.org/Team%3AUNILA_LatAm/Communication)[Funding](https://2021.igem.org/Team%3AUNILA_LatAm/Funding)
* Team

  [Team Members](https://2021.igem.org/Team%3AUNILA_LatAm/Team)[Attributions](https://2021.igem.org/Team%3AUNILA_LatAm/Attributions)[Collaborations](https://2021.igem.org/Team%3AUNILA_LatAm/Collaborations)[Partnership](https://2021.igem.org/Team%3AUNILA_LatAm/Partnership)[Sponsors](https://2021.igem.org/Team%3AUNILA_LatAm/Sponsors)
* Awards

  [Medal Criteria](https://2021.igem.org/Team%3AUNILA_LatAm/Medal_Criteria)

# Experiments

---

##### Contents

# Introduction

For the Synfronteras team, participating in the iGEM competition represents a unique opportunity to learn and collaborate with synthetic biology, establishing laboratory conditions that allow us to develop projects on wetlab and making synthetic biology a traditional line of research at our university. Nonetheless, the pandemic of COVID-19 caused a sanitary and economic crisis in the whole world. In these exceptional conditions and aiming to achieve our goals, it was essential to think about the safety of the team members who would attend the laboratory.

In Brazil, the vaccination against SARs-CoV-2 started later than countries from Europe and North America. Therefore, our team only carried out specific activities until the availability of vaccines. When the vaccine became available, the team members that were fully vaccinated performed most laboratory activities.

# Team goals

To develop the project on wetlab, the team counted on the collaboration of associate professors and researchers from our university and others around the country. These collaborations were crucial for obtaining plasmids and microorganisms that would be essential in our project and valuable to standardizing protocols and procedures. The initial activities were focused on the: preservation of microorganisms, obtaining purified plasmids and chemo-competent bacteria, transformation efficiency tests and other basic molecular biology procedures.

# Project goals

Due to our limitations and the unusual conditions caused by the pandemic, the main goal of the team was to demonstrate the parts of our design that were considered essential to the project. In this sense, we chose to test our elimination module, whose main feature is using of a trypsin-activated effector molecule, antimicrobial peptide (AMP) DRS-N of the dermaseptin class, that might induce toxicity in *Leishmania spp.* [1]. For this, it was necessary to obtain proAMP, test its activation through trypsin-induced proteolytic cleavage and evaluate the leishmanicidal activity of AMP

# Work cell

Initially, to test our elimination module, it was necessary to produce our effector molecule. Although *Bacillus subtilis* is more suitable for AMP production, due to the unavailability of an electroporator and the difficulty of transforming *B. subtilis* by other transformation methods (eg. heat shock), the bacterium *Escherichia coli* was chosen as the host to obtain proAMP [2]. Furthermore, *E. coli* also presents advantages related to ease of cultivation and chemo competency induction, crucial factors in view of the limited time available for laboratory experiments.

# Assembly

Knowing that we would use Gibson assembly to assemble our parts, we designed our gBloks to be as modular as possible, first we selected primers suitable for use in our expression plasmid pBBR1MCS-2 and cloning plasmid pUC19, to produce common ends. We used benchling platform to generate overlap regions, which was the same for both plasmids, so we used the same overlap in the design of all our inserts. Additionally, we standardized to put biobrick prefix and suffix sequences before each overlap to enable biobrick assembly.

![The primers designed can be used to linearize both our expression and cloning plasmids, and as it's in the lacZ region it is possible to be used in other plasmids.](https://static.igem.org/mediawiki/2021/f/f3/T--UNILA_Latam--fig1.png)

Figure 1: The primers designed can be used to linearize both our expression and cloning plasmids, and as it's in the lacZ region it is possible to be used in other plasmids.

![The overlaps designed were used in all insert fragments and, this way, can all be assembled in both linearized plasmids or others who used the previously designed primers.](https://static.igem.org/mediawiki/2021/d/d9/T--UNILA_Latam--fig2.png)

Figure 2: The overlaps designed were used in all insert fragments and, this way, can all be assembled in both linearized plasmids or others who used the previously designed primers.

All parts designed maintained a modular character and were divided into two modules, the expression/export module and the coding module, allowing its use in different contexts except for the main (G1) and reporter (G0) circuits, designed for a specific objective.

![Our module of expression/export and the proAMP module can be freely combined among themselves.](https://static.igem.org/mediawiki/2021/d/d8/T--UNILA_Latam--fig3.png)

Figure 3: Our module of expression/export and the proAMP module can be freely combined among themselves.

After obtaining the linear pBBR1MCS-2, we used the NEBuilder HiFi DNA Assembly Cloning Kit to assemble the G1 and G0 parts and cloning in NEB 5-alpha Competent *E. coli* (C2987H), and reclone at *E. coli* BL21 (D3). The protocols used for this experiment, P006A, P006B, P008A, P009A, P010A, P011A, P002A, can be found on our [protocol page](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols).

To confirm the assembly, we chose used PCR to amplify the inserts and the electrophoresis to visualize it, using as template plasmid miniprep and colonies from selection plates.

# ProAMP obtainment

To produce our proAMP, we used the expression strain *Escherichia coli* BL21 (D3), which is inducible by IPTG, and the expression vector pBBR1MCS-2, with our main circuit (G1) T7-LacO Promoter + RiboJ + RBS\_0034 + 6xHis-tag + proDRS-N1 + double Terminator (BBa\_K4075008).

After the production, we carry out the obtention of the effector molecule by collecting the extracellular portion of the cultivation and the periplasmic extract of cells. The protocols used for this experiment, P012A, P013A, can be found on our [protocol page](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols).

# Trypsin proteolytic cleavage

According to Guilherme Brand, responsible for the characterization of DRS-N1, the propeptide is a trypsin-like target, which is evident when we observe the cleavage site between the acidic piece and the AMP. Nonetheless, there are other spots in the AMP that are susceptible to trypsin action, as shown:

![The cleavage sites were predicted by the peptide cutter tool from ExPASy. Both KR amino acids are in the propeptide section and are more likely to be cleaved by trypsin; furthermore, there are other cleavage sites, which indicates the possibility of partial digestion.](https://static.igem.org/mediawiki/2021/c/ca/T--UNILA_Latam--fig4.png)

Figure 4: The cleavage sites were predicted by the peptide cutter tool from ExPASy. Both KR amino acids are in the propeptide section and are more likely to be cleaved by trypsin; furthermore, there are other cleavage sites, which indicates the possibility of partial digestion.

We believe that for this characteristic, the activation of the proAMP may occur by partial digestion, so the amount of trypsin will be determinant for the AMP activity. We treated the extracellular and periplasmic obtention products with three different concentrations of trypsin 0.5, 1.0 and 1.5 mg/mL to test this aspect. According to previous studies using trypsin digestion, these concentrations were chosen[3][4]. After that, the final step of our experiments allows us to evaluate which conditions are ideal for the AMP activation.

Partial digestion can be an advantage knowing that the trypsin inside the sand-fly midgut is not fully available due to its role in digesting the blood meal. **Protocol used:** [P014A](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols)

# Leishmanicidal assay

To the final step of our experiments, we verified the leishmanicidal activity of our AMP and how the trypsin treatment worked on activating it. For this purpose, cultures of *Leishmania infantum* were incubated with the products derived from the bacterial culture portion and the periplasmic extract of cells, both previously treated with trypsin. The *Leishmania sp.* were observed after five days of incubation. **Protocol used:** [P015A](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols).

# Future Perspectives

For 2021 participation on iGEM, the only aspect tested in wetlab were characteristics from the elimination module. However, there are several questions still to be solved and demonstrated. Many studies are still needed once this project uses a complex implementation strategy in a new application.

Besides testing the elimination module, it would be needed to verify the remaining modules on wetlab, to demonstrate engineering success and complete the engineering design cycle (Design + Build + Test + Learn + Design) on these modules too.(To see the modules check our [design page](https://2021.igem.org/Team%3AUNILA_LatAm/Design)). For the detection module, the major issue was the characterization of endogenous miRNAs to test the feasibility of the designed toehold switch. Its function could be demonstrated by synthesizing the miRNAs candidates in a second circuit evaluating the activation of the switch.

On the Protection device module, it would be necessary to induce the chassis to its sporulated form and test it in the presence of L-alanine, which would turn it for its vegetative form, making it available for detection and expression of other circuits.

The Killswitch module requires an optogenetic tool to expose the chassis to light, as, after transitioning to its vegetative form, our killswitch system becomes sensitive to expression triggers. This way, we could obtain quantitative data on the light exposure needed to kill the bacteria.

Beyond testing the modules, it would be necessary to validate the chassis, using a closed system to deliver it transformed with a reporter gene to sand-fly in sugar baits. In the next step, a sample of the population tested would be evaluated to see if *Bacillus subtilis* could colonize sandfly midgut.

Our team believes that answering these questions could help enlighten the pathways of paratransgenesis through synthetic biology and, hopefully, this work will be continued, synthesizing a new future.

# References

1. Brand, G. et al. (2013). The Skin Secretion of the Amphibian Phyllomedusa nordestina: A Source of Antimicrobial and Antiprotozoal Peptides. Molecules 2013, Vol. 18, Pages 7058-7070, 18(6), 7058-7070. <https://doi.org/10.3390/MOLECULES18067058>
2. Ji, S. et al. (2017). Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700. Scientific Reports 2017 7:1, 7(1), 1-10. <https://doi.org/10.1038/srep40587>
3. KAWASAKI et al. (1986) et al. Limited Digestion of Cahnodulin with Trypsin in the Presence or Absence of Various Metal Ions. The Journal of Biochemistry, v. 99, n. 5, p. 1409-1416. <https://academic.oup.com/jb/article-abstract/99/5/1409/1011673>
4. VISHWESHWARAIAH et al. (2021)Rational design of hyperstable antibacterial peptides for food preservation. npj Science of Food, v. 5, n. 1, p. 26. <https://www.nature.com/articles/s41538-021-00109-z>

A Project by

![](https://static.igem.org/mediawiki/2021/d/dc/T--UNILA_LatAM--LogoV3.png)

Contact

synfronteras@gmail.com

Sponsor

[![](https://static.igem.org/mediawiki/2021/8/89/T--UNILA_LatAM--LogoIPH.png)](https://www.itaipu.gov.br/)

Follow us!

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# Team:UNILA LatAm/Results

Results | iGEM UNILA\_LatAm

[![](https://static.igem.org/mediawiki/2021/9/98/T--UNILA_LatAm--Logo.png)](https://2021.igem.org/Team%3AUNILA_LatAm)

* Project

  [Description](https://2021.igem.org/Team%3AUNILA_LatAm/Description)[Design Overview](https://2021.igem.org/Team%3AUNILA_LatAm/Design_Overview)[Background](https://2021.igem.org/Team%3AUNILA_LatAm/Background)[Engineering](https://2021.igem.org/Team%3AUNILA_LatAm/Engineering)[Contribution](https://2021.igem.org/Team%3AUNILA_LatAm/Contribution)
* DryLab

  [Design](https://2021.igem.org/Team%3AUNILA_LatAm/Design)[Model](https://2021.igem.org/Team%3AUNILA_LatAm/Model)
* WetLab

  [Experiments](https://2021.igem.org/Team%3AUNILA_LatAm/Experiments)[Results](https://2021.igem.org/Team%3AUNILA_LatAm/Results)[Notebook](https://2021.igem.org/Team%3AUNILA_LatAm/Notebook)[Protocols](https://2021.igem.org/Team%3AUNILA_LatAm/Protocols)[Parts](https://2021.igem.org/Team%3AUNILA_LatAm/Parts)[Safety](https://2021.igem.org/Team%3AUNILA_LatAm/Safety)
* Human Practices

  [Introduction](https://2021.igem.org/Team%3AUNILA_LatAm/Human_Practices)[Sustainability](https://2021.igem.org/Team%3AUNILA_LatAm/Sustainability)[Implementation](https://2021.igem.org/Team%3AUNILA_LatAm/Implementation)[Fighting Underreporting](https://2021.igem.org/Team%3AUNILA_LatAm/Fighting_Underreporting)[Integrated Human Practices](https://2021.igem.org/Team%3AUNILA_LatAm/Integrated_Human_Practices)[Education](https://2021.igem.org/Team%3AUNILA_LatAm/Education)[Inclusivity](https://2021.igem.org/Team%3AUNILA_LatAm/Inclusivity)[Communication](https://2021.igem.org/Team%3AUNILA_LatAm/Communication)[Funding](https://2021.igem.org/Team%3AUNILA_LatAm/Funding)
* Team

  [Team Members](https://2021.igem.org/Team%3AUNILA_LatAm/Team)[Attributions](https://2021.igem.org/Team%3AUNILA_LatAm/Attributions)[Collaborations](https://2021.igem.org/Team%3AUNILA_LatAm/Collaborations)[Partnership](https://2021.igem.org/Team%3AUNILA_LatAm/Partnership)[Sponsors](https://2021.igem.org/Team%3AUNILA_LatAm/Sponsors)
* Awards

  [Medal Criteria](https://2021.igem.org/Team%3AUNILA_LatAm/Medal_Criteria)

# Results

---

##### Contents

# Introduction

Our team understands that demonstrating the project's applicability as a whole would be ideal for presenting a suitable proof of concept. However, due to our limitations and the unusual conditions caused by the pandemic, our goal was to demonstrate the parts of our design that we considered critical for the project. The elimination module is responsible for *Leishmania spp.* inactivation, which is essential for the Biopank functionality. Therefore we attempt to demonstrate it. The description of each step is available on our [experiments page](https://2021.igem.org/Team%3AUNILA_LatAm/Experiments).

# Assembly

At first, we struggled a little to obtain our expression plasmid pBBR1MCS-2 linearized. Before getting the Monarch Miniprep Kit (New England Biolabs, USA), we carried out an alkaline **lysis** miniprep, however did not have RNase to clean up the RNAs. After using the miniprep kit, a cleaner result was obtained, but as the plasmid is a low copy one, our yields were not high.

After the obtention of the purified plasmid, PCR reactions for linearization were started. Still, due to a low yield of DNA fragments on the gel and the lack of a proper DNA ladder, we could not be sure of having a positive result, leading us to digest lambda DNA and use it as a ladder. All the steps were repeated to get the purified linear plasmid. Later, we realized that the lower yield could be related to the melting temperature used, since there was a difference between the Tm calculator temperature and the one provided by NEB Tm calculator.

![Lanes three to seven results from different PCR reactions to linearize the pBBR1MCS-2 plasmid. The first lane with the EcoRI digested lambda DNA worked as a ladder since the available one was impracticable. Using literature information about lambda DNA, we could estimate the molecular weight of fragments, therefore concluding the successful linearization of the plasmid for its size of 5148 bp.](https://static.igem.org/mediawiki/2021/a/af/T--UNILA_Latam--Result1.png)

Figure 1: Lanes three to seven results from different PCR reactions to linearize the pBBR1MCS-2 plasmid. The first lane with the EcoRI digested lambda DNA worked as a ladder since the available one was impracticable. Using literature information about lambda DNA, we could estimate the molecular weight of fragments, therefore concluding the successful linearization of the plasmid for its size of 5148 bp.

We successfully assembled the two constructs selected in our experiments: the G1 containing the CDS of the proAMP and G0, with the reporter gene of mCherry. The assembly was confirmed in *E. coli* DH5-alpha by PCR with miniprep and colonies from selection plates amplifying the inserts followed by electrophoresis.

![The first lane with the product PCR from the G0 synthesis (gBlock) amplification is a positive control, leading us to find the positive samples at lanes 2-3 and 9-10, from E. coli DH5-alpha, assembled with G1. Those samples were obtained by PCR amplification of miniprep and colony PCR. The difference between the positive control and positive samples is justified because G0 is 1179 bp long and G1, 614 bp.](https://static.igem.org/mediawiki/2021/2/2f/T--UNILA_Latam--Result2.png)

Figure 2: The first lane with the product PCR from the G0 synthesis (gBlock) amplification is a positive control, leading us to find the positive samples at lanes 2-3 and 9-10, from E. coli DH5-alpha, assembled with G1. Those samples were obtained by PCR amplification of miniprep and colony PCR. The difference between the positive control and positive samples is justified because G0 is 1179 bp long and G1, 614 bp.

The miniprep from *E. coli* DH5-alpha was used to clone *E. coli* Bl21(D3). The cloning confirmation was done by colony PCR amplifying the inserts followed by electrophoresis. Additionally, other samples from *E. coli* DH5-alpha assembled with G0 were picked to be confirmed. All the assemblies were cultivated and preserved.

![This new conformation electrophoresis used the same positive control. The G0 assembly on E. coli DH5-alpha was confirmed as positive on the third lane. At lanes four to six, colony PCR from E. coli BL21 transformed with G1 assembly was confirmed as positives. A linear sample of pBBR1MCS-2 was used as a negative control.](https://static.igem.org/mediawiki/2021/1/10/T--UNILA_Latam--Result3.png)

Figure 3: This new conformation electrophoresis used the same positive control. The G0 assembly on E. coli DH5-alpha was confirmed as positive on the third lane. At lanes four to six, colony PCR from E. coli BL21 transformed with G1 assembly was confirmed as positives. A linear sample of pBBR1MCS-2 was used as a negative control.

# proAMP production

The expression of our proAMP is inducible by IPTG, and it has the peptide signal OmpA [1]. A specific timing of induction and cultivation conditions can favor the exportation of the proAMP to the periplasmic and extracellular region, this would save some time and facilitate the purification process[2][3]. We planned to identify the production of the AMP by immunoblot using the 6xHis-tag as target, but unfortunately we got our parts later than expected and **couldn't perform it**.

In this shorter time to present results, we decided to obtain our effector molecule from extracellular and periplasmic media for an indirect demonstration through he inactivation of *Leishmania infantum*.

# AMP activation and leishmanicidal activity

The leishmanicidal assay was incubated for five days, under the six samples of activated AMP from the last step and control samples, including proAMP without enzymatic treatment. We expected to count *Leishmania infantum* viable cells to determine their growth during the incubation time. Unfortunately, the medium used wasn't in good condition to properly cultivate the cells, and contamination was found. Due to these factors, the experiment was interrupted.

It took longer than expected to get the gene synthesis and assemble the parts, so there was no more time to restart the experiment and get results before the wiki freeze. We hope to present these results later in 2021 or at upcoming participation.

# References

1. Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of *B. subtilis* chitosanase enzyme from E. coli expression system. SpringerPlus, 5:1200, 2016. [Article.](https://springerplus.springeropen.com/articles/10.1186/s40064-016-2893-y)
2. Peng, L., Xu, Z., Fang, X., Wang, F., & Cen, P. (2004). High-level expression of soluble human Î²-defensin-2 in *Escherichia coli.* Process Biochemistry, 39(12), 2199-2205. doi:10.1016/j.procbio.2003.11.011. [Article.](https://www.sciencedirect.com/science/article/pii/S0032959203004333?casa_token=UFlHYNhqcA8AAAAA:f7itQtkbLJcnCghtS0WG6Ypt2VUcQiWbmCy2QXC1-k2r5EJH0gMim4QynqDVa9gIbU-SM74nxJLlSw)
3. Rollan, C H. et al. Protein expression and extraction of hard-to-produce proteins in the periplasmic space of *Escherichia coli.* Technical University of Denmark, 2020. dx.doi.org/10.17504/protocols.io.bdr2i58e. [Protocol.](https://www.protocols.io/view/protein-expression-and-extraction-of-hard-to-produ-bdr2i58e?step=8)

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