Team:Ecuador/Notebook
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Training in the principles of synthetic biology
course in edX. Participation:
Everyone
Training in the principles of synthetic biology
course in edX. Participation:
Everyone
Training in the principles of synthetic biology
course in edX. Participation:
Everyone
Participation and collaboration in the
introduction workshop to synthetic biology dictated by Heber Torres in
Universidad de las Fuerzas Armadas ESPE. Participation: Everyone
February
- Week 17
●
Meeting to choose the project that will participate in iGEM 2018. Participation:
Everyone
Brainstorm project ideas about synthetic biology
PROJECTS:
-
Spider silk production in yeast
-
Production of ivory in yeasts to reduce the furtive hunting by
this material
-
Production of colicine in Saccharomyces
boulardii to use it as a
probiotic antibacterial infections
-
Recombinant production of fusion proteins and their coupling to
bacterial cellulose for obtaining a biomaterial.
February
- Week 18
●
Meeting to decide the winning project. Participation: Everyone
The winning project was: “Recombinant production of fusion proteins and their coupling to
bacterial cellulose for obtaining a biomaterial”
Meeting with two researchers with experience in
synthetic biology and Igem competitions and they help
us to delimit the project. Participation:
Everyone
March
- Week 20
In this week, we designed the logo and the name
of our project. Participation:
Everyone
THE WINNER!!!
March
- Week 21
●
Looking for sponsoring with Ecuadorian enterprises but we only get
the sponsorship of two: IDgen and Tuki.
Participation: Estefany
Paredes and Camila Velandia
Dragon Tatoo!!
●
We decided to make a raffle to raise funds for the registration in
iGEM 2018. Participation:
Everyone
March
- Week 22
●
In this week the winners of the raffle were awarded. Participation: Everyone
●
We also chose the design of our shirts. Participation: Everyone
●
Inscription in Igem 2018 with funds
obtained
Videoconference with Heber Torres to elucidate
the best methods to follow for the development of the project. Participation: Everyone
April
- Week 24
Videoconference with the Tec de Monterrey to
discuss our project. Participation: Estefany Paredes, Camila Velandia,
Jennifer Tapia, Nicolas Lopez.
April
- Week 25
Interview with channel “Teleamazonas”
to spread the synthetic biology. Participation:
Everyone
April
- Week 26
●
Participation in the mathematical modeling course dictated by
delft university of technology in edX platform.
●
Participation in dynamical modeling methods for systems biology in
Coursera platform, dictated by Icahn school of medicine at Mount Sinai. Participation: Estefany
Paredes in collaboration with Francisco Mosquera.
This week we dedicated ourselves completely to
finish the two online courses we were doing. Participation: Estefany Paredes in
collaboration with Francisco Mosquera.
May
- Week 28
Review of topics learned in online courses and
search parameters. Participation: Everyone
May
- Week 29
●
Start of sequence design
●
Sequence design for cellulose production. Participation: Everyone
May
- Week 30
Sequence design for the
production of fusion proteins. Participation:
Everyone
We dedicate this week to the search of
parameters for a modeling. Participation:
Everyone
June
- Week 32
Meeting with the Ministry of Environment for
construction of the country position in synthetic biology, digital sequences
and risk assessment and management guides for GMO. Participation: Nicolas Lopez in collaboration with Francisco Mosquera.
June
- Week 33
●
Start of the Interlab. Searching the plate reader with absorbance and
fluorescence in others universities of Ecuador.
Biomedicine team of University "UTE" let us use their equipment,
where Linda Guaman, our instructor, works. Participation: Natalia Torres in
collaboration with PI’s and Instructors.
●
Video conferencing with the Estonia iGem
team
June
- Week 34
●
Interlab:
Preparation of materials, LB broth and agar plates with chloramphenicol (25 ìL/mL), SOC media, SOB agar, CCMB80 Buffer. Participation: Natalia Torres and Jennifer Tapia
●
Realization of modeling. Participation:
Estefany Paredes in collaboration with Francisco Mosquera.
●
Meeting up Latinoamérica
●
Interlab:
Preparation of stock solutions, application and measurement of calibration
protocols. Participation: Natalia
Torres, Juan Carlos Luzuriaga in collaboration with
Eduardo Moncayo.
July
- Week 36
●
Application in the national unpublished financing program
●
Interlab:
Application of competent cells protocol and practice transformation protocol of
iGEM. In the photo, you can see a control of the
transformation protocol with RFP. Participation:
Juan Carlos Luzuriaga and Jennifer Tapia in
collaboration with Eduardo Moncayo.
July
- Week 37
Interlab: Transformation with the BioBricks
and measurement of fluorescent cells. In the photo, you can see the laboratory
of Biomedicine team in the University "UTE". Participation: Jennifer Tapia, Juan Carlos Luzuriaga
and Natalia Torres in collaboration with Eduardo Moncayo.
July
- Week 38
●
Interlab:
Repetition of transformation with the BioBricks and
measurement of cells. Application of Protocol: Colony Forming Units per 0.1
OD-- E. coli cultures. Sending of raw data and forms. Participation: Jennifer Tapia, Juan Carlos Luzuriaga
and Natalia Torres in collaboration with Eduardo Moncayo.
●
Conducted talks on the importance of synthetic biology in the
world and prospective in the country, this talk was held in 4 schools in Quito:
Colegio de Liga, Liceo policial, Émile Jaques
Dalcroze, Ángel Polibio Chávez. Participation:
Everyone.
●
Competent cells preparation
●
Células E. coli DH5α, BL21(DE3) y
BL21(DE3) pLyss were prepared using the CaCl2 method,
modifying slightly the one described in: http://parts.igem.org/Help:Protocols/Competent_Cells. Participation: Estefany Paredes in
collaboration with Francisco Mosquera.
●
Resuspention of gBlocks:
bscA, bscB, bscC1, bscC2, bscD, bsCcPax and bsCmCax; with 20μl dH20, to reach a concentration of
50 ng/ìL. Participation: Camila Velandia
●
Domestication with overhang PCR of bscA,
bscB, bscC1, bscC2 and bscD.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained expected bands of bscA (Lane 2 - 2536 bp), bscB (Lane 3 - 2827 bp) and bscC2
(Lane 5 - 2139 bp); and unspecified bands of bscC1
(Lane 4) and bscD (Lane 6). Participation: Juan Carlos Luzuriaga in
collaboration with Eduardo Moncayo.
●
Linealization of the psb1C3 and psb1A3 plasmids
with PCR, using 8% DMSO. Amplification of LacI from
the commercial plasmid pET-24a-ELP[V-150] (Addgene).
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained expected bands of
psb1C3 plasmid (Lane 2), psb1A3 plasmids (Lane 3) and LacI
(Lane 4 - 1420 bp). Participation: Jennifer Tapia in collaboration with Eduardo Moncayo.
●
Domestication of the gBlocks (CBD cipA, BMP2, sfGFP). With the objective that the restriction enzymes EcoRI and PstI can cut gBlocks forming cohesive ends, we perform a PCR to add
their recognition sites. But in the design, we did not know that there should
be base pairs that flanked a restriction site, therefore, the first test
failed, but with the proper investigation we discovered this observation, then
sent to request primers to add those bases in prefix and subfix.
●
In this week, we also collaborated with the TEC CEM team
participating in a musical video about igem. Link: https://youtu.be/R7vdiGLLJZA?t=319. Participation: Estefany Paredes in
collaboration with Francisco Mosquera.
August
- Week 41
●
Domestication with overhang PCR of bscD
and LacI.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained unspecific bands (Lane
2) and expected bands of bscD (Lane 3 and 4 - 854 bp) with an unspecific band, and LacI
(Lane 5, 6 and 7 - 1420 bp). Participation: Juan Carlos Luzuriaga in
collaboration with Eduardo Moncayo.
●
This week came the primers that we ordered
and we amplified and purification the gBlocks: CBD cipA under the control of the promoter LacI,
BMP2 and sfGFP. Participation:
Estefany Paredes in collaboration with Francisco Mosquera.
NOTES:
Gel de agarosa al 1%
1X
TAE Buffer
Lane
1: 1kb DNA ladder
Lane
2: PCR product of BMP2 gBlock (541 bp)
Lane
3: PCR product of CBD cipA gBlock
(806 bp)
Lane
4: PCR product of sfGFP gBlock
(928 bp)
Gel de agarosa al 1%
1X
TAE Buffer
Lane
1: 1kb DNA ladder
Lane
2-5: PCR products of elastin-like polypeptide V150.
Lane
6: Purified PCR product of CBD cipA gBlock
Lane
7: Purified PCR product of BMP2 gBlock
Lane
8: Purified PCR product of sfGFP gBlock
●
We decided to carry out a molecular techniques course in order to disseminate synthetic biology tools. Participation: Everyone
August
- Week 42
● Transformation (on the left CBD cipA-BMP2 and to the right is the CBD_sfGFP) Participation: Estefany Paredes in collaboration with Francisco Mosquera.
●
Construction of the composite part cbd cipA-ELP. We obtained expect brands of PCR ELP product from
pET-24a-ELP[V-150] using the primers Forward 1 y Reverse1 with 509 pb. Participation:
Estefany Paredes in collaboration with Francisco Mosquera.
Notes:
Lane 1: 100 pb
DNA ladder
Lane 2: PCR ELP product from pET-24a-ELP[V-150] using the
primers Forward 1 y Reverse1
Lane 3: PCR ELP product from pET-24a-ELP[V-150] using the
primers Forward 1 y Reverse3
Lane 3: PCR ELP product from pET-24a-ELP[V-150] using the
primers Forward 1 y Reverse2
●
Domestication with overhang PCR of bscC1.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane A), we obtained an expected band of
bscC1 (Lane B - 2152 bp) with two unspecific bands. Participation: Nicolas Lopez
August
- Week 43
●
We obtained a similar pattern of bands, 500 bp,
this is because it is a repetitive sequence so we
decided to purify the fragment obtained and then sequence it. Participation: Estefany
Paredes in collaboration with Francisco Mosquera.
NOTES:
Gel de agarosa al 1%
1X TAE Buffer
Lane 1: 100 pb
DNA ladder
Lane 2: Purified ELP550
Lane 3: No sample
Lane 3: Unpurified ELP550
●
Domestication with overhang PCR of bsCcPax.
Participation: Natalia Torres in
collaboration with Eduardo Moncayo.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained unspecific bands (Lane
2-6).
●
Talks on the importance of synthetic biology in the world and
prospective in the country, in Unidad Educativa
Antares. Participation: Diego Garzon
and Natalia Torres, in collaboration with Francisco Mosquera.
●
In this week, we obtained the results of the sequence that was
sent to sequence.
●
Domestication with overhang PCR of bsCmCax
and bsCcPax.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained unspecific bands of bsCmCax (Lane 2) and expected band of bsCcPax
(Lane 3 - 1512 bp), but with low concentration. Participation: Natalia Torres in
collaboration with Eduardo Moncayo.
●
Domestication with overhang PCR of bscA,
bscB and bsCmCax.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained expected bands of bscA (Lane 2 - 2536 bp), bscB (Lane 3 - 2827 bp) and bsCmCax (Lane 4 - 1522 bp). Participation: Natalia Torres in
collaboration with Eduardo Moncayo.
●
We prepare the shipment of our parts and send the plasmids
psb1c3-CBD cipA-BMP2 y psb1c3-CBD cipA-sfGFP. Participation: Camila Velandia in collaboration with Francisco Mosquera.
●
Gibson assembly. Participation:
Nicolas Lopez in collaboration with Eduardo Moncayo.
In the electrophoresis gel
(1% agarose) with 1 kb DNA ladder (Lane 1), we obtained degraded DNA in another
Lanes. We faced with several problems that occurred during this trial and we
could not obtain the assembled vectors that we wanted.
●
We prepare the shipment of our parts and send the plasmids
psb1c3-CBD cipA-BMP2 y psb1c3-CBD cipA-sfGFP. Participation: Estefany Paredes and Camila Velandia, in collaboration with Francisco Mosquera.
●
Extraction
of fusion protein CBD-sfGFP with
●
Domestication with overhang PCR of bsCcPax.
Participation: Nicolas Lopez
In the electrophoresis gel (1% agarose) with 1
kb DNA ladder (Lane 1), we obtained expected band of bsCcPax
(Lane 2 - 1512 bp) with two tenuous unspecific bands.
●
In
this week, we started with the characterization of the binding strength of the
CBD cipA with an endogenous C-terminal linker.
Through fluorescence measurement of: cellulose with GFP like negative control,
cellulose with the fusion protein CBD-sfGFP and the
last one, after three washes with differents fluids:
blood plasma, PBS, saliva and distilled water. Participation: Natalia Torres and Estefany
Paredes, in collaboration with Francisco Mosquera.
●
Gibson assembly. Using the Gibson assembly kit instructions from
NEB with DMSO, we obtained the same result of first Gibson assembly. We believe
that there are loops forming during the process. Participation: Nicolas Lopez in collaboration with Eduardo Moncayo.
●
A third course of
molecular techniques was carried out. Participation:
Everyone
October
- Week 49
●
Finishing the wiki. Participation:
Everyone
●
Celebration of Birthday of our PI Francisco Flores. Thanks for
everything!!! Participation: Everyone.
October
- Week 50
Meeting with the Undersecretary of Natural
Heritage of the Ministry of the Environment. Presentation of the iGEM Ecuador Project to facilitate the understanding and
future linkage of Modern Biotechnology, in the creation of norms and public
policies. Participation: Diego
Garzon and Nicolas Lopez.
