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TEC-CEM | Notebook

ITESM-CEM | Enzy7-K me

Notebook 3401



July 2013

Team iGEM TEC CEM was assembled on July 24. A total of 12 persons, from different areas and backgrounds were selected to take a part in this project. Team captain Carlos Meza organized meetings with all the members in order to explaining what iGEM was and the theoretical bases of the competition.

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August 2013

Brainstorming. Each team member shared different propositions for the project, with different approaches to a variety of subjects. Afterwards, individually or in pairs, several ideas were explored more thoroughly, with the intention to consolidate the theorical basis, experimental development and applications. All of this was done taking the igem requirements into account.

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September 2013

We approached several members of the faculty here at Tec CEM, to see if they wanted to be our advisors, based on the needs of the different projects. Finally, we selected Dr. Edgar García (PH.D. in Biotechnology) M.C. Ana Laura Torres (Master in Sciences), and Dr. Aurora Antonio (PH.D. in Enzimology).

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October 2013

Time to develop the brainstormed ideas obtained on August

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November 2013

Time to develop the brainstormed ideas obtained on August

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December 2013

We were ready to choose our project.

  • December 13. After a couple months of brainstorming, we finally exposed our project candidates to our advisors, Dr. edgar, m.c. ana laura, and dr. aurora; our career director Dr. Berenice Vergara, and the Biotechnology and Chemical Engineering Department director, Dr. Josefina Castillo. During this session, said advisors helped us choose by making remarks regarding the experimental development, viability, and potential applications for every project. At the end, 3 candidates were selected for further examination, in order to ensure that the project was an adequate proposal for a competition of this caliber.

  • December 15.A second meeting was scheduled in order to make the final decision. Voting was done to choose the best project. Lipofuscine degradation was the chosen one. *Dramatic music* During the holiday break, we all had to do research in order to come back in January with all set up for the project.

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January 2014

After thorough considerations during the holiday break, it was deemed that the project would have better viability with cholesterol degradation instead of lipofuscine degradation. This way, there was a new approach for the project, and the application was set on medical biorremediation for the treatment of artheroschlerosis.

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February 2014

Work groups were assigned

  • The whole february.Since laboratory work would be carried out in the summer, work groups were assigned in order to cover other aspects of the project, such as sponsorships, mathematical modelling, human practices, in silico analysis of the biobricks and so forth. Throughout this period each group worked individually and result reports were given out during the weekly meetings.

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March 2014

In spite of the division in work groups, every memeber of the team carried out research, in order to ensure that we all had the theoretical bases and that we all deeply knew what the project was about. During this month, Mario Dominguez was helping the team with the theory regarding the mathematical modelling, which was to be carried out alongside the experimental development.

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April 2014

Having adequate theoretical bases, final desing of the plasmid began, while taking into account the necessary biobricks. This way, a general protocol for the summer work could be drafted. Sponsorship search began and contact was established with different corporations.

  • April 5.First human practices: DNA extraction and synbio for kids.

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May 2014

Lets start Wet Lab!

  • May 11.Design of primers to obtain new Biobricks.

  • May 21.BioBricks arrived! Now lab work could begin.

  • May 24.First day of the second activity of human practices; Synthetic Biology Course.

  • May 31.Second day of Synbio course.

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June 2014

Lab-work keeps going!

  • Week 1. / of June was the last and third day of Synbio Course

  • Weak 2.During this week, the first PCR assays were carried out under different conditions, in order to find the optimal ones. Previously extracted plasmid pcDNA myc his 3.1. Optimal conditions were found for CMV and NeoR.

  • Weak 3.PCR assays were carried out under different conditions, in order to find the optimal ones. Previously extracted plasmid pcDNA myc his 3.1. Optimal conditions were found for Ori and BGHPA.

  • Week 4. We were preparing the third human practices activity; Medical Biotechnology Course. Having July 21 as the first day. Work with the iGem kit started. The different mediums and cultures were prepared in order to correctly carry out the transformations. After several tests, the correct antibiotic concentration was found for each biobrick. Each team member was assigned with a transformation, in order to leard how to use the kit and get ahead with the work.

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July 2014

Lets use iGEM Plates!

  • Week 1. After LB plaques grew with the different transformations, the bacteria were regrown in liquid medium in order to ensure presence of the plasmids. 2 cultures were made out of each colony, in order to avoid loss of any transformed colony whatsoever.

  • Week 2. During this week, plasmid extraction in order to have available plasmids from different colonies and to confirm the presence of our BioBricks of interest. Electrophoresis was carried out in order to ensure correct plasmid extraction. Now we could carry out the next step: digestion

  • Week 3.Summer break!

  • Week 4. To confirm the presence of our wanted BioBricks, enzymatic digestion was carried out with different restriction enzymes on the plasmids. Samples were run in gel and this way the presence of the BioBricks was confirmed. Once this was done, cryopreservation of what was left of the colonies was done in order to have them available for posterior usage.

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August 2014

3 months to go!

  • Week 1. • Successful PCRs of BGHPA and NeoR were achieved with NEB® PCR Supermix (25µl each).
    • Cryopreservation and extraction of B0017 and B0015 parts.
    • Digestion of B0015 part.
    • Successful PCR of F1Ori (25 µl) achieved with NEB® PCR Supermix.
    • Successful PCR of CMV (25 µl) achieved with NEB® PCR Supermix.
    • Digestions of B0017 and B0015.
    • During this week we also tried a new protocol for the transformation of pPROEX plasmids in dH5α by thermal shock. We also started to work with cholesterol oxidase and oxidoreductase, two of the three enzymes we’re going to assembly for the final part of the project by a previous transformation of PUC57 in dH5α by thermal shock as well; both transformations were positive. After the incubation, the isolated colonies of the transformations in dH5α, were again inoculated in LB petri dishes with ampicillin 35 mg/ml. We also applied the digestion-ligation protocol to prove the biobrick was present in the transformed plasmids.

  • Week 2. • Successful PCRs of BGHPA (3*50 µl) achieved with NEB® PCR Supermix.
    • Successful PCR of F1Ori (3*50 µl) and CMV (3*50 µl) achieved with NEB® PCR Supermix.
    • Purification of the PCR products F1Ori and BGHPA with Phenol-Chloroform.
    • Successful PCRs of NeoR (3*50 µl) achieved with NEB® PCR Supermix.
    • We verified in an electrophoresis that the various PCRs have been amplifying successfully except NeoR, which has some unspecific amplifications, the annealing temperature will be raised from 62ºC to 62.5ºC.
    • The purifications with Phenol-Chloroform show a dim line in the electrophoresis, we suspect of an ill stored Phenol-Chloroform (ambient tº and it shows a pink color). • After the growth of the isolated colonies in the petri dishes, we inoculated them in liquid LB with ampicillin for a further plasmid extraction of cholesterol oxidase, oxidoreductase and pPROEX; in which we tried a different time of incubation and time for the denaturalization of the lysozyme to prevent the appearance of genomic DNA. We also transformed dehydratase, the third enzyme, in dH5α by thermal shock, which then was subjected to plasmid extraction under the standards of the modified protocol.

  • Week 3. • Successful PCRs of NeoR (3*50 µl) achieved with NEB® PCR Supermix, this time a unique band appeared in the electrophoresis.
    • A new Phenol-Chloroform was prepares and stored correctly (refrigerated and inside an opaque glass container).
    • Purification of F1Ori, BGHPA and CMV PCRs with Phenol-Chloroform.
    • We wanted to do the first digestion and ligation of our bioparts, nevertheless the Phenol-Chloroform extractions weren’t satisfactory so we’ll have to wait and learn another technique to purify PCR products.
    • A purification of the BGHPA PCR product was made with ethanol and we helped the other groups in our team. • During this week, we tried a ligation with K823005 and E0240 in PSB1C3. We inoculated on a dish plate, and then isolated twelve colonies to identify the different possibilities of ligation of each biocrick; then were ran an electrophoresis to prove their right ligation. Only one out of the twelve colonies was correctly ligated. We also inoculated dehydratase, oxoacylreducatse and cholesterol oxidase in 50 ml of liquid LB with ampicillin 35 mg/ml for a further plasmid extraction and expression in an alternative plasmid.

  • Week 4. • The purification of BGHPA was satisfactory, so we proceeded to digest with EcoRI and SpeI.
    • We kept on helping the other groups in our team preparing various Cam+ mediums.
    • Further attempts at purifying PCR products with ethanol were not successful. So we decided to keep on helping our partners until we learn a new purification protocol from our tutor.
    • Purifications of NeoR, CMV, BGHPA and F1Ori were made with a new protocol.

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September 2014

Project in sight! and Viva México!

  • Week 1 • During these days, and after the assurance of the correct enzyme ligations with the vector, we ligated each enzyme with B0034, a RBS that would be useful for a further expression in animal cells. These enzymes are going to be overexpressed and purified.
    • We ran an electrophoresis to make sure are PCR purifications weren't contaminated and we confirmed they weren't.
    • We digested the 4 bioparts with EcoRI and SpeI.
    • We did a ligation of the bioparts.
    • Transformations of the ligations of BGHPA and F1Ori.
    • Ligation of CMV with J04450.
    • The transformations weren’t successful so they were repeated.
    • Ligations of BGHPA and F1Ori with J00450.
    • New transformations of the BGHPA and f1ori ligations were made since they haven’t been successful.
    • Colonies of the transformation of PproexB were cultured in new dishes and essay tubes.
    • An essay tube with the CMV ligation showed growth so it was cultured in Cam+ dishes.
    • The Cam+ dishes with CMV showed a pink colored growth when we were expecting white cultures, new dishes were cultured from the original essay tube.
    • We helped our companions in extractions and digestions of other bioparts.

  • Week 2 • We kept on helping our companions with digestions and ligations.
    • In the morning our CMV culture finally showed the expected white color in a culture, so we proceeded to grow a new dish from this culture.
    • We prepared multiple essay tubes with Amp+ and Cam+ and petri dishes with Cam+ for the growth of cells transformed by `both us and our companions.
    • At night the CMV culture had grown so we cultured it in 50ml of LB/Cam+.
    • We also cultured at night 1ml of PproexB in 50ml of LB/Amp+.
    • The CMV+J00450 plasmid was extracted and digested in order to verify it, it showed an unexpected pattern in its bands, another enzyme will be used for its verification.
    • A ligation of BGHPA+J00450 was made.
    • The ligation of F1Ori+J00450 will have to wait since all the PstI enzyme we need in order to digest J00450 has all been used, we’ll need to acquire more enzyme.
    • We still haven’t acquired PstI so our advance is halted by this inconvenient.
    • We transformed cells with the BGHPA+J00450 ligation.
    • The BGHPA+J00450 transformed cultures showed a white coloration (at least three different cultures).
    • We were able to digest J00450 with PstI in buffer 3.1.
    • A ligation of F1Ori+J00450 was made.

  • Saturday September 13 – Tuesday September 16, 2014. No lab activities to report because of a long weekend. (Independence day)

  • Week 3 • The CMV+J00450 ligation was verified with XhoI, the pattern of the bands obtained was, once again, unexpected, a new gel electrophoresis will be made with a lower voltage and a longer running time.
    • F1Ori was digested again, since the previous ligation was lost in the weekend.
    • The original F1Ori ligation was found so yesterday’s digestion was stored.
    • The F1Ori+J00450 ligation was transformed.
    • SOC media was prepared.
    • The F1Ori+J00450 transformation was unsuccessful.

  • Week 4 • We made a digestion of NeoR with SpeI and EcoRI. • We made a digestion of BGHPA with PstI and EcoRI. • We made a digestion of J00450 with SpeI and EcoRI for its later ligation with NeoR. • Ligation of NeoR+psB1C3. • We transformed NeoR. • We cultured the NeoR+psB1C3 transformation in LB media. • The NeoR+ transformation was extracted.

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October 2014

Just one last step!

  • Weeks 1,2,3 & 4.During these final weeks, we started overexpressing the proteins, as pPROEX has an inducible promoter with IPTG. For each enzyme we made a growth curve starting from inoculation at time zero, and ending six hours after the induction with IPTG (right after the optical density was between 0.5-0.6). We took samples each hour from the time zero to the end of the curve. Those samples were prepared with Laemmli buffer for a further analysis by SDS-PAGE to identify the protein and to see the overexpression of the same, the phase where the protein was accumulating (soluble phase or inclusion bodies) and the isolation of the sample for purification by affinity chromatography.
    NeoR, CMV, and BGHPA characterization. Our enzyme substrates where delivered out of time so we couldn't characterize them. Time matters :(
    We have submitted our new biobricks to iGEM HQ.
  • See you in Boston!
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