Team:TecCEM/Notebook
Notebook
13 - 19 May.
Summary
Research for characterization began and sub-projects deadlines were assigned. Logistics of the Latin American Meet-up began. A visit to Six Flags was made.
Wet lab
Preparation of solutions to be used for the competent cells. Transformation efficiency for E. coli DH5α and antibiotic viability were tested.
Dry lab
Delimitations of the mathematical model were made. Pitching of software ideas and sub-projects. We started looking into the possibility of creating a skinchip in a mini reactor. Investigation about the scaffold heterotrimer was appointed. The development of biobricks for recombinant leptin and collagen was started. A meeting with an expert in miRNA therapy was appointed, new insights about anti-miRNA were established from that meeting and the viability was researched. Leptin and collagen were codon optimized for their recombinant production in Escherichia coli BL21 (DE3) and Rosetta (DE3) respectively.
Human Practices
Committee for human practices was delimited. A brainstorming of ideas was done and the best ideas were picked up in order to develop them in the near future.
20 - 26 May.
Summary
Worklab began and we had meeting to assign new sub-project. Wiki page planification began, we contacted a Graphic designer to help out. We also contacted several specialists in tissue regeneration to gain insights about the project.
Wet lab
Lab got closed off for maintenance reasons. The iGEM kit got stuck at the customs border protection.
Dry lab
Skinchip 3D model was designed and software ideas were pitched. Biobricks were checked and revised. Chemical modifications for anti-miRNA were investigated, locked nucleic acid (LNA) technology appointed as the best method for miRNA silencing. Receptors within the scaffold for stem cell migration/call was investigated but also other molecules apart from the main project protein/molecules were investigated for the same purpose.
Human Practices
Fundraiser organization and planification, tasks were handed out. Went to a public talk about tissue regeneration in burn victims using nanoparticles of chitosan, we made contact with the people involved in the conference and received a feedback, another meeting with the experts was appointed. We had a meeting with a communication reporter to be published in a magazine. Online meet up with iGEM Brazil, Ecuador and Chile were performed.
27 May - 2 June.
Summary
Worklab began and we had meeting to assign new sub-project. Wiki page planification began, we contacted a Graphic designer to help out. We also contacted several specialists in tissue regeneration to gain insights about the project.
Wet lab
Transformation of competent E. coli DH5 cells was performed using different plasmids, no successful transformation was seen. New cells were created under the same protocol. Escherichia coli DH5 transformed with psB1C3 and inoculated in LB medium. A Skin chip prototype was manufactured using PDMS and normal objects such as clips and shelf sustainers.
Dry lab
Protein sequences for the ECM were optimized with IDT and verified. Novedous methods for the miRNA silencing were analyzed theoretically and several critical factors for the competitive binding against RISC complex were encounter. Physico-chemical characteristics for leptin were revised and documented. Several designs were proposed for the modelling and manufacturing of the skin chip.
3 June- 9 june.
Summary
We had a meeting with Doctor Pablo Rodriguez from the Special Center for burn children of the General Hospital of Toluca Dr. Nicolás San Juan.
Wet lab
We transformed E.coli DH5 alpha with non important plasmids, in order to verify efficiency. Also alkaline plasmid extraction were done tested with electrophoresis.
Dry lab
Biobricks were revised. Chitosan and leptin union to scaffold protocol was designed. We sent our sequences to IDT for synthesis. Skinchip design was created and defined an operational model.
10 June- 16 june.
Summary
This week we recorded our video for a fundraiser. The Latin American Meetup logistic is being planned and organized. We had pizza on friday.
Wet lab
We re-tested alkaline plasmid extraction protocols and reviewed it with electrophoresis. Cell culture area was sterilized for HaCAT cell line subculture. Antimicrobial peptide LL37 from Trieste team contained in iGEM Plate Distribution 2017 was transformed in DH5-alpha.
Dry lab
Chitosan protocol revised and changed after a meeting with an expert. iGEM’s kit is still stuck at the customs border. The docking between proteins was modeled. A skinchip model was designed and revised with several experts.
Math model
The math model for pharmac diffusion along a matrix was investigated, the equation of Weilbull was chosen and variables for leptin were investigated.
17 June- 23 june.
Summary
This week a lot of progress was made, we had a meeting with Doctor Gerardo Manuel García Lozano of Foundation RinoQ hospital. We had courses that dived into tissue and cell culture and the correct lab safety cautions that need to be taken from Laboratorios de Especialidades Inmunológicas SA de CV (LEI), a laboratory that specializes in immunology and tissue cultures. Our Interlab Kit was greenlighted by the customs border!
Wet lab
HaCAT subculture was made. L929 cell line was established and given the correct maintenance and subcultures were made. Primers for VF2 and VR were obtained and Agilent Assembly Kit primers designed and. We dried plasmids, in order to send it to iGEM team TecChihuahua. A PCR using iGEM consensus for LL37 was performed.
Dry lab
The skinchip was modeled in 3D and the bioreactor workflow chart created. Research is being revised and protocols created for lab work.
Human practice
Several arrangements were performed with RinoQ foundation to coordinate the trip to visit childrens that have suffered of burns and they helped us by giving feedback about the project. A visit with Fernando Osobampo, a specialist in plastic surgery, that has had a lot of close contact with the recovery of people that had suffered of burns, we had a lot of feedback and focused our project in second degree burns and had an approval of the efficiency of our delivery method.
24 June- 30 june.
Summary
This weekend we continue with the realization of the characterization of LL37. Several advances were created in order to organize the first main human practice of the team. Hardware and mathematical models were finally design. A visit with a specialist in plastic surgery was realized. The iGEM distribution kit arrived at our laboratory (26/07/18).
Wet lab
The primers for Agilent kit were resuspended in order to create workable stocks (10 M). A enzyme restriction was performed to analyze the LL37 sequence amplified last week. The Agilent kit reactants were aliquot into workable stocks as the protocol indicated. Chitosan first nanoencapsulation was performed using a negative control (water) and a lipase for encapsulation.
Dry lab
Agilent Sure Vector kit protocol was read.
1 July- 7 July.
Summary
On this weekend the laboratory was mostly closed because of maintenance and the visit to RinoQ was done. The characterization of LL37 continued by making the first step to the assembly protocol for Agilent kit.
Wet lab
A PCR using the agilent synthesized primers was performed, in order to attach the necessary sequences for the overlap used in the assembly of the plasmid.
Human practice
A visit to RinoQ foundation, in San Luis Potosí, was achieved. An assessorament with experts in the burns area was done to make the project more efficient. A coordination between TecCEM and RinoQ was created, in order to create an application to help burn victims and to prevent burns.
8 July- 14 July.
Summary
We had the iGEM Latin America 2018 Meet Up! We had the pleasure to receive teams from all over Mexico and Latin America such as Brazil, Ecuador, and Chile where they presented their projects and got feedback from specialists. This Meetup was important for the teams to get an overall look into their work and something to note is that the event got a lot of diffusion online. We also started the planification and logistics for Universium as next week the event would take place.
Wet lab
The first try to do an assembly of LL37 was performed using the previous LL37 fragment and a PCR cycle with all the reactants of the Agilent kit. Successfully after the second try the plasmid was assembled with the LL37 sequence, this was done by increasing the number of cycles to 16. This assemble gave us a plasmid that contain a T7 promoter, bacterial Ori, Amp resistance, terminator and the LL37 protein along with a His tag.
Human practice
The iGEM Latin America Meet Up had place in our campus, we had the presence of teams from Monterrey, Guadalajara, Chihuahua and virtual presence of teams from Brazil, Chile and Ecuador. In this meet up the teams had the opportunity to give a presentation such as pre-run to practice the one that has to be done in Boston, in addition several specialists were there to analyse and give feedback in the projects. The logistics of the visit to Universium was performed.
15 July- 21 July.
Summary
We had meetings with several teams seeking if we could collaborate with them and see how the rest of the iGEM teams are doing to try and expand communication around the scientific community. We also went on with the preparations for Universum. We continued with the characterization of LL37 and the transformation of E. coli. The DNA synthesize from IDT was delivered and the first project insights could be achieved. This week Interlab was done in the lab to obtain the results.
Wet lab
The plasmid with LL37 was transformed in E. coli strains as Rosetta gami and BL21 (DE3), the transformations were successful as single colonies were achieved. In order to verify the transformation plasmid extraction was performed and positive results were obtained. The IDT sequences for collagen type V (COL5), tenascin domain V (TCD5) and leptin (LEP) were resuspended in bio mol water for the creation of stocks. A ligation to the plasmid psB1C3 was performed by enzymatic restriction and T4 DNA ligation.
Human practice
The visit to Universum was done in the weekend, during this time we explained biotechnology concepts to children and gave them a little insight to the subject. We also talked with their parents to explain more about the science field in genetic engineering and the project in general terms, by doing this we expected that biotechnology and the general public can come into contact and become a subject of interest for everyone. In addition, we took this opportunity as a way to gather some data by doing a survey, which will help us in the development of an app for burn prevention.
22 July- 28 July.
Summary
This week we finished Interlab and sent the forms to iGEM, we continued as well to communicate with other iGEM teams from around the world to collaborate and we started to organize with them to do the video. We advanced with the transformation of the iGEM parts in E. coli strains and with the design of our hardware, but also we started seeking for the establishment of an experiment which could asses our project viability.
Dry lab
In order to evaluate our project we started the searching of a protocol which we could use in our human cell lines to recreate the effects caused by burn accidents with either hot liquids or hot surfaces, which was told by the foundation Rhino Q to be the most common ones. We obtained two main articles which had a lot of potential to our project, both articles described the methodology in which we could treat our cell lines to recreate this burning effect and also it was described how we could measure it. By those means our assays would involved the usage of a hot glass Petri dish and the evaluation of cell viability by the usage of MTT or other molecules.
Wet lab
We transformed E. coli DH5 alpha for plasmid multiplication purposes resulting into correct results for BBLEP and BBTNC5, for BBCOL5 the observance of red colonies were determined as a non conclusive result because of the problem that BBCOL5 part construction had a constitutive RFP as a reporter but the re-ligation of any iGEM plasmid also results in a RFP but controlled by a lac promoter, in order to verify the results obtained a posterious induction / non induction experiment must be done to determine the veracity of the ligation. We made a quick trial into the recreation of burn assay into our cellular lines, we obtained interesting results showing that the exposure of 15, 30 and 60 seconds to PBS at approximately 80oC showed a decrease in cell morphology and cell viability, the results were obtained by cell counting in Neubauer chamber with trypan blue and microscope visualization.
Hardware
We came to a possible proposal of a skin chip and designed a 3D model including the mechanisms, the proper calculations and how to automate the system. This model still very naive and is needed to get proper correction in order to create the first design of the skin chip.
Interlab
The parts of iGEM kit plate distribution was transformed in E. coli DH5 alpha strain and alkaline plasmid extraction was performed and agarose gel analysis was done to verify plasmid existence. Calibration and cell measurement protocols were performed and submitted in the wiki (https://2018.igem.org/Team:TecCEM/InterLab).
29 July- 4 August.
Summary
This week was the last week of summer break so we had to work harder than ever. A briefing about what was done during the summer was broken down in a meeting and a new planification of objectives was done.
Wet lab
We performed the transformation of all project parts (BBCOL5, BBLEP and BBTNC5) into protein production strains such as BL21(DE3) and Rosetta (DE3). After alkaline plasmid extraction and analysis in electrophoresis we could conclude that the existence of the parts project BBLEP and BBTNC5 were achieved, even though several more analysis must be performed in order to obtain significant results. In the case of BBCOL5 an experiment of induction/non induction was performed to analyse if the ligation product was a re-ligation of iGEM plasmid, the results obtained concluded that the part was indeed a re- ligation product and for so on BBCOL5 ligation was discarded as part of the project but used as a medium to obtaining RFP for nanoencapsulation experiments.
Hardware
We contacted COMSOL for their software specialized in microfluidics also a professor in our campus that specializes in microfluidic modelling was contacted for tutoring.
5 August- 11 August.
Summary
This weekend we started classes in our university, also we had significant advances in the verification of plasmid existence (project parts) in bacteria colonies. We started production and purification of protein of interest. We had an incident in the lab too, our centrifuge broke and the cellular lines died!
Dry lab
We tried to make a custom medium broth for collagen production and consulted with a professor to reassure the calculous sheet was correct but the idea was shut down because it could have had too many variables.
Wet lab
We started with protein production of protein LL37 of E. coli strains: Rosetta, DH5a and BL21. The electrophoresis analysis in polyacrylamide gel of LL37 protein production was performed but no results were achieved, this could have been due to lysis protocol, thus new purification protocols were revised. The cellular lines of HaCaT and L929 had died because of a runout accident of CO2 master tank, producing acidification and loss of morphology, plans for obtaining new aliquots of cellular lines started. The device BBCOL5 was tried to ligate into plasmid pSB1A3, but no consistent results were achieved.
12 August- 18 August.
Summary
This week we had several team meetings to get some team building going and continued the protein production for characterization. Also the first advances of a complete hardware were finally achieved.
Wet lab
New protocols for purification of LL37 protein were done. RIPA buffer lysis protocol was used but polyacrylamide gel analysis was not achieved because of difficulties in visualization. We improved our polyacrylamide protocol and got a new Coomassie solution given as a gift from one professor which really helped us to visualize our gels more easily. For protein production analysis of the project parts (BBLEP and BBTNC5), an induction curve was performed, in which samples of each device transformed into BL21(DE3) were analyzed in a time lapse of 9 hours after IPTG induction, taking aliquots of 1 mL per hour for posterious protein analysis experiments, each aliquot was done by duplicated and observed in spectrophotometer at OD600 to correlate protein production with total biomass. Also we obtained new HaCaT cell lines.
Math model
We had a meeting with several professors to help us get a better idea about the whole math model and the reach our project could have.
Hardware
Models for skin chip were design using AutoCad and Catia softwares, the modelations of flows were done and the materials needed were investigated.
19 August- 25 August.
Summary
This week we had several advances into the software and hardware part of the project. The protein curve production was analysed by polyacrylamide gel. The first insights into RFP purification were obtained.
Wet lab
The protein curve was analyzed by polyacrylamide gel and the results obtained were not good enough, several factors affected the quality of the gel. We started with nano encapsulation of RFP and visualization of nanoparticles in SEM microscoped.
26 August- 1 September.
Summary
Our protein production began, we consulted a professor specialized on proteins that gave us several tips on it. We reworked the hardware design as a final one that is less expensive and more innovative, then we sent the design into a company so the corp was made.
Wet lab
We created a new production protein curve and performed an analysis by electrophoresis and Bradford quantification. The polyacrylamide gel was done at 20% to obtain a better separation of protein, although no significant overproduction bands were seen. The quantification by Bradford methodology showed a low quantity of protein in pellet centrifugation. This results made us re-evaluate the lysis protocol and change the quantification methodology. We made contact with a professor expert in recombinant protein production and we obtained new crucial points for a new definitive curve. We made nano encapsulation with RFP and saw it in SEM microscope, no structure was seen, we started seeking for new methodologies for obtaining better resolution in the results. We performed a new assay of in vitro skin burn in L-929 cell line.
Hardware
The manufacturing of the skin chip was investigated, the final material used for this purpose was glass as it can be reused and can help labs to cut their dependence in trash-able cell culture bottles. Several companies were contacted in order to quote prices, and at the end the company “” was contacted to create the skin chip carcass.
2 September - 8 September.
Summary
On this week we retried the curve of protein production of BBLEP and BBTNC but applying the points that our professor gave us, the results obtained made us replan the project methodology into a fresh beginning.
Wet lab
After the results obtained last week we set the objective to prove that our proteins could be produced by the bacteria. For this to be achieved we made a new production protein curve with different new settings such as: BL21(DE3) with another plasmid to corroborate that bands seen were not result of antibiotic resistance peptides, creation of a non induced protein curve, and starting induction until OD600 of 0.7. The results obtained were not significant and there were no bands showing our proteins. Also we performed a PCR to an alkaline extraction of plasmids coming from BL21(DE3) transformed strains (BBLEP and BBTNC), using VR and VF2 primers, the results obtained show no amplification of BBLEP and inconsistent results for BBTNC. With that on mind we aimed to redo the transformation of parts and protein production, in order to obtain new and much better results. We also obtained kanamycin for the ligation of BBCOL into psB1K3. We realized the quantification of lactate of our burned cell lines at 0.5, 1, 2 and 3 minutes, in order to get numerical data that will translate into proliferation rate after a burn accident, those results will help us into developing a more accurate math model for the purposes of the project.
Dry Lab
We started to investigate new lysis protocol to ensure that our protein was released from the bacterial pellet, we obtained one protocol which was specific for the purification of tag His proteins derived from BL21(DE3), but also we attended with our professor specialist in protein purification for new protocols, she gave us a Laemmli buffer for fast visualization of results for protein induction. Also we received the idea from one of our assessors to use RIPA buffer for the lysis, but after an extensive internet research no methodology was seemed to work with E. coli pellets.
Hardware
We went to CDMX to obtain the hoses and syringes for the assembly of the skin chip. Also we talked with professor Chong to land the idea of how we will be measuring the proliferation rate in the skin chip, the idea ended up in the usage of Tsc3200, which is a programmable converter of light (colour) to raw data, that will measure the virage (acidification) of DMEM medium from rose into orange, which is due to the metabolism of cells and would be a good parameter for account how much the cell is growing given the application of the project and when those the system must change the medium.
9 September - 15 September.
Summary
This week we re-organized the project objectives according to a our new chronogram and replanificated our biggest goals to achieve the production and purification of our proteins, also nanoparticles creation was performed. We started our production from ground zero as we were not having any positive results, and as a way to troubleshoot this we made new competent cells to produce our proteins. Nonetheless, the hardware prototype was finally obtained and some tests were performed to get insight into it’s functionality.
Wet lab
The production and purification of RFP was successful by implementing the new lysis buffer, even though, a modification in the buffer must be performed to increase the lysis efficiency given that there was still red color in the pellet. Using the resultant RFP we started the nano encapsulation, the result was store at 4oC. We started the creation of completely new BL21 (DE3) competent cells for the posterious transformations of newly ligated parts. We obtained kanamycin for the implementation of psB1K3 in the ligation of BBCOL5 part. We performed the transformation of the new BL21 (DE3) chemocompetent cells and we obtained white colonies, by that mean, we performed induction of protein expression in the new colonies to completely verify the presence of our proteins, for a posterious polyacrylamide gel analysis. Sadly, after the incomplete subculture of L929 given by the loss of functionality of our trypsin, no good cells could be incorporated into the new flask and because of that we lost our cell line, for so on we must obtain another aliquot.
Dry Lab
We researched a new lysis protocol for purification of proteins. The protocol we obtained was specific for cell lysis and nickel affinity purification of E. coli BL21. From that protocol we keep special attention in the lysis buffer which is comprised of many reductant agents. Also in an article which described the lysis method for E. coli we found that his lysis buffer was exactly the same as the one we previously encounter, for that we decided to give that buffer a try but with modifications according to our experience such as incubation with lysozyme.
Hardware
We obtained the prototype of the “skin reactor” and performed a test to determine exhaust capacity of the prototype using only inclination and centrifugal force. The results obtained showed that an angle of 7.5° was necessary to remove most of the medium from the medium chamber. For that the creation of a base would be necessary for the removal of medium given those characteristics. We then proceed to modelate the base in CATIA and later we 3D printed it in our school.
16 September - 22 September.
Summary
Great news ! On this week we started gazing the first possible obtention of our proteins. Also we created more important components for the transformation of BBCOL5. The hardware was improved by incorporating all the components necessary for his functionality.
Wet lab
For the nanoparticles we then went to IPN institute to use their TEM (transmission electron microscope) and NanoSight to show the results obtained, we could proudly say that the first extraction of RFP nanoparticles was achieved and a good image from TEM was obtained.
Hardware
The final assemblies were done and the automatization parts were laser cut for their incorporation.
23 September - 29 September.
Summary
This week we began working way harder in the lab as deadlines approach. We decided as a team the wiki order and design, as well as a name for our hardware.
Wet lab
Nanoparticles standardisation was done with Bradford assay for a quick encapsulation once Leptin begins production. PBS pH 7.4 was used to recreate physiological medium and test nanoparticles liberation.
Hardware
The last assemblies were done and the sensors were tested and connected.
30 September - 6 October.
Summary
As deadlines approach, we looked into the option of synthesizing new primers to fix a DNA sequence problem in our Biobricks. The Wiki Freeze being so close we started uploading all our results to our wiki. We had a lot of stress going on and ran the final tests on our project. We were both exhausted and excited to reach the conclusion of this great experience as a team.
Wet lab
Nanoparticles were observed in Nanosight with the encapsulation of BSA to confirm the size. Some liberation assays were done to quantify protein concentration with PBS at pH 7.4 to simulate physiological medium. For the parts, a PCR was done. Some gels were run. A sequencing company was contacted to troubleshoot our sequence.
Software
The software design was defined and most of the databases completed.