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.