iGEM 2018 — Team Ecuador

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Ecuador/Notebook

[iGEM ECUADOR](https://2018.igem.org/Team%3AEcuador)

[PROJECT](https://2018.igem.org/Team%3AEcuador/Description)

[DESCRIPTION](https://2018.igem.org/Team%3AEcuador/Description)
[BACKGROUND](https://2018.igem.org/Team%3AEcuador/Background)
[MODELING](https://2018.igem.org/Team%3AEcuador/Model)
[DESIGN](https://2018.igem.org/Team%3AEcuador/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3AEcuador/Experiments)
[NOTEBOOK](https://2018.igem.org/Team%3AEcuador/Notebook)
[RESULTS](https://2018.igem.org/Team%3AEcuador/Results)

[PARTS](https://2018.igem.org/Team%3AEcuador/Parts)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AEcuador/Public_Engagement)
[BEST INTEGRADED HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Best-In-Human-Pract)

[SAFETY](https://2018.igem.org/Team%3AEcuador/Safety)

[INTERLAB](http://2018.iGEM.org/Team%3AEcuador/Interlab)

[JUDGING FORMS](http://2018.iGEM.org/Team%3AEcuador/Judging)

[TEAM](https://2018.igem.org/Team%3AEcuador/Team)

[TEAM MEMBERS](https://2018.igem.org/Team%3AEcuador/Team)
[COLLABORATIONS](https://2018.igem.org/Team%3AEcuador/Collaborations)

[ATTRIBUTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)

[ATRIBUTTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)
[SPONSORING](https://2018.igem.org/Team%3AEcuador/Sponsoring)

C-lastin, Interlab

# NOTEBOOK

### [Index](#INT)

* [November2017](#l1)
* [December 2017](#l2)
* [January 2018](#l3)
* [February 2018](#l4)
* [March 2018](#l5)
* [April 2018](#l6)
[* May 2018](#l7)
[* June 2018](#l8)
[* July 2018](#l9)
[* August 2018](#l10)
[* September 2018](#l11)
[* October 2018](#l12)

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Nov 17** | **Dec 17** | **Jan**  **18** | **Feb**  **18** | **Mar 18** | **April 18** | **May 18** | **June 18** | **July 18** | **Aug 18** | **Sept 18** | **Oct 18** |
| 1 | 6 | 10 | 15 | 19 | 23 | 27 | 31 | 35 | 39 | 44 | 48 |
| 2 | 7 | 11 | 16 | 20 | 24 | 28 | 32 | 36 | 40 | 45 | 49 |
| 3 | 8 | 12 | 17 | 21 | 25 | 29 | 33 | 37 | 41 | 46 | 50 |
| 4 | 9 | 13 | 18 | 22 | 26 | 30 | 34 | 38 | 42 | 47 | 51 |
| 5 |  | 14 |  |  |  |  |  |  | 43 |  |  |

**November
2017**

Training in the principles of synthetic biology
course in edX. **Participation:**
Everyone

![](https://static.igem.org/mediawiki/2018/a/a4/T--Ecuador--1NovJan.jpeg)

**December
2017**

Training in the principles of synthetic biology
course in edX. **Participation:**
Everyone

![](https://static.igem.org/mediawiki/2018/a/a4/T--Ecuador--1NovJan.jpeg)

**January
2018**

Training in the principles of synthetic biology
course in edX. **Participation:**
Everyone

![](https://static.igem.org/mediawiki/2018/a/a4/T--Ecuador--1NovJan.jpeg)

**February
-** Week 16

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”**

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/s2048x2048/43335395_1707385856056269_4035039667702202368_n.jpg?_nc_cat=105&oh=aff81ed89ff132b1780a50113efba827&oe=5C4798EE](https://static.igem.org/mediawiki/2018/f/ff/T--Ecuador--18.jpeg)

**March
-** Week 19

Meeting with two researchers with experience in
synthetic biology and Igem competitions and they help
us to delimit the project. **Participation:**
Everyone

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43231354_1197354867081256_677067788526813184_n.jpg?_nc_cat=104&oh=2d91b18c9aed66ff8f6753feb69c116d&oe=5C5802AC](https://static.igem.org/mediawiki/2018/c/c7/T--Ecuador--19.jpeg)

**March
-** Week 20

In this week, we designed the logo and the name
of our project. **Participation:**
Everyone

![](https://static.igem.org/mediawiki/2018/d/d8/T--Ecuador--N20.jpeg)![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43258895_547933522304534_3709188521953591296_n.jpg?_nc_cat=105&oh=2d6ef0cef10ebe88a39fd4f126856329&oe=5C15EE59](https://static.igem.org/mediawiki/2018/5/5a/T--Ecuador--N20.2.jpeg)

THE WINNER!!!

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43306425_1435063509929921_4156025207053615104_n.jpg?_nc_cat=102&oh=791a79548927506602fbd240dec43885&oe=5C5F0BFD](https://static.igem.org/mediawiki/2018/3/3c/T--Ecuador--N20.3.jpeg)

**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

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/43250603_2149175432011317_5588531103896961024_n.jpg?_nc_cat=101&oh=9d84b267413373b24b73b020e581aa77&oe=5C3ED801](https://static.igem.org/mediawiki/2018/5/52/T--Ecuador--N21.jpeg)![La imagen puede contener: texto](https://static.igem.org/mediawiki/2018/2/2d/T--Ecuador--N21.2.jpeg)

**Dragon Tatoo!!**

●
We decided to make a raffle to raise funds for the registration in
iGEM 2018. **Participation:**
Everyone

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/s2048x2048/43360908_245161822838177_3574548229301207040_n.jpg?_nc_cat=107&oh=cfdd2904bfc4fdb9358bebfaf22caeaa&oe=5C3DB213](https://static.igem.org/mediawiki/2018/a/a8/T--Ecuador--N21.3.jpeg)

**March
-** Week 22

●
In this week the winners of the raffle were awarded. **Participation:** Everyone

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43534293_554365394995957_2453547209401040896_n.jpg?_nc_cat=108&oh=894ea5d2447aabe4bbe72838535bfe8a&oe=5C549ED2](https://static.igem.org/mediawiki/2018/5/59/T--Ecuador--N22.jpeg)

●
We also chose the design of our shirts. **Participation:** Everyone

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/44112017_179591562958241_7047730802305531904_n.jpg?_nc_cat=105&oh=05d00f150cb8c6676e3b5a8157eca12d&oe=5C5F4C39](https://static.igem.org/mediawiki/2018/2/22/T--Ecuador--N22.2.jpeg)

●
Inscription in Igem 2018 with funds
obtained

**April
-** Week 23

Videoconference with Heber Torres to elucidate
the best methods to follow for the development of the project. **Participation:** Everyone

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43308185_2157869347867067_8340309207265837056_n.jpg?_nc_cat=107&oh=eb60606b1cf5ec87bba77ab58290c3f3&oe=5C505D06](https://static.igem.org/mediawiki/2018/e/e5/T--Ecuador--N23.jpeg)

**April
-** Week 24

Videoconference with the Tec de Monterrey to
discuss our project. **Participation:** Estefany Paredes, Camila Velandia,
Jennifer Tapia, Nicolas Lopez.

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43221054_351351188938589_765933307580907520_n.jpg?_nc_cat=106&oh=b7347106399ad3dad59662d330d45bc2&oe=5C4DBDED](https://static.igem.org/mediawiki/2018/c/c2/T--Ecuador--N24.jpeg)

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/43293037_1626139370823675_8290006412856131584_n.jpg?_nc_cat=111&oh=3475c2df7d953aaf3664b8cdcca7df9b&oe=5C4D65F0](https://static.igem.org/mediawiki/2018/7/73/T--Ecuador--N24.2.jpeg)

**April
-** Week 25

Interview with channel “Teleamazonas”
to spread the synthetic biology. **Participation:**
Everyone

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43302776_361654364576437_8285811690096820224_n.jpg?_nc_cat=107&oh=527a7b7943bcc5182e74ac6f3e1e9224&oe=5C45A4B3](https://static.igem.org/mediawiki/2018/c/ce/T--Ecuador--N25.jpeg)

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43224565_331449260765902_4281315952903061504_n.jpg?_nc_cat=110&oh=acc9d4b50128283b6b04fcfba87a63d9&oe=5C144442](https://static.igem.org/mediawiki/2018/a/aa/T--Ecuador--N25.2.jpeg)

**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.

**May
-** Week 27

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

**June
-** Week 31

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.

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43236071_146861622933895_7023530169126092800_n.jpg?_nc_cat=100&oh=9c7cb29438f74247b90fe416be3b54e5&oe=5C5E4BDC](https://static.igem.org/mediawiki/2018/5/53/T--Ecuador--N32.jpeg)

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43425694_165956737670133_8428731163572436992_n.jpg?_nc_cat=105&oh=2d81fea2e0cdb2e4392aff32f7b96e71&oe=5C18B3B7](https://static.igem.org/mediawiki/2018/3/3f/T--Ecuador--N32.2.jpeg)

**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

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43246154_688905561483201_4986756325452021760_n.jpg?_nc_cat=111&oh=d3adb054bcbe6c6f892f2f1969e8092b&oe=5C48F8B1](https://static.igem.org/mediawiki/2018/6/63/T--Ecuador--N33.jpeg)

**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.

**July
-** Week 35

●
Meeting up Latinoamérica

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/43239826_1361615433970879_8244643715600089088_n.jpg?_nc_cat=108&oh=97e606758d6b3c33558315cdcb6cf766&oe=5C629FE6](https://static.igem.org/mediawiki/2018/4/49/T--Ecuador--N35.jpeg)

●
**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

![](https://static.igem.org/mediawiki/2018/6/61/T--Ecuador--N36.jpeg)

●
**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

![](https://static.igem.org/mediawiki/2018/6/68/T--Ecuador--N37.jpeg)

**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.

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43200653_326900648085192_6687097001349218304_n.jpg?_nc_cat=103&oh=38f8290692b683bfb04ed736b09baad3&oe=5C62E0A1](https://static.igem.org/mediawiki/2018/c/cb/T--Ecuador--N38.jpeg)

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43301688_321050321807845_4335225605013372928_n.jpg?_nc_cat=105&oh=71f00df7fdfc349379e0f6e7e3ac2a6f&oe=5C43FB6B](https://static.igem.org/mediawiki/2018/9/99/T--Ecuador--N38.2.jpeg)

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43221152_765588607110001_6553308666999603200_n.jpg?_nc_cat=105&oh=4a13f9c326eaed4ae16250a154657d1e&oe=5C5213E6](https://static.igem.org/mediawiki/2018/3/3c/T--Ecuador--N38.3.jpeg)

![](https://static.igem.org/mediawiki/2018/e/ea/T--Ecuador--N38.4.jpeg)

**August** - Week 39

●
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](http://parts.igem.org/Help%3AProtocols/Competent_Cells). **Participation:** Estefany Paredes in
collaboration with Francisco Mosquera.

![https://scontent.xx.fbcdn.net/v/t1.15752-0/p280x280/43662693_266801410637666_283709777043783680_n.jpg?_nc_cat=111&_nc_ad=z-m&_nc_cid=0&oh=4f1eb5b2b0f6b1b393b20f6fca3127a6&oe=5C5DE8F7](https://static.igem.org/mediawiki/2018/1/17/T--Ecuador--N39.jpeg)

●
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.

![](https://static.igem.org/mediawiki/2018/9/90/T--Ecuador--N39.2.jpeg)

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.

**August** - Week 40

●
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).

![](https://static.igem.org/mediawiki/2018/0/09/T--Ecuador--N40.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/2/28/T--Ecuador--N41.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/8/8f/T--Ecuador--N41.2.jpeg)

**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)

![](https://static.igem.org/mediawiki/2018/3/35/T--Ecuador--N41.3.jpeg)

NOTES:

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

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/43348589_1913390148707583_6336327902753193984_n.jpg?_nc_cat=107&oh=da27691abf3f02d149c1d854fe091439&oe=5C1AB9CD](https://static.igem.org/mediawiki/2018/2/2e/T--Ecuador--N41.4.jpeg)

![https://scontent-arn2-1.xx.fbcdn.net/v/t1.15752-9/s2048x2048/43335410_1140173299464654_4058855325783031808_n.jpg?_nc_cat=105&oh=14c641f6fe3c548a8c23b80fa648d2aa&oe=5C54BD8A](https://static.igem.org/mediawiki/2018/6/69/T--Ecuador--N41.5.jpeg)

**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.

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/44219843_294636631264524_4433553413761925120_n.png?_nc_cat=104&oh=dc86c354b46db956b6d6f16bfc53cd3c&oe=5C605337](https://static.igem.org/mediawiki/2018/2/2e/T--Ecuador--N42.jpeg)

●
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.

![](https://static.igem.org/mediawiki/2018/a/a9/T--Ecuador--N42.2.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/5/57/T--Ecuador--N42.3.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/7/7a/T--Ecuador--N43.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/5/52/T--Ecuador--N43.2.jpeg)

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.

![](https://static.igem.org/mediawiki/2018/f/f9/T--Ecuador--N43.3.jpeg)

**September -** Week 44

●
In this week, we obtained the results of the sequence that was
sent to sequence.

![https://scontent.fgye7-1.fna.fbcdn.net/v/t1.15752-9/43562255_302119250384716_6644610029510983680_n.jpg?_nc_cat=108&oh=4196c45b3bf1f3ec3c99df16b648d9d0&oe=5C4F4E52](https://static.igem.org/mediawiki/2018/6/6b/T--Ecuador--N44.jpeg)

●
Domestication with overhang PCR of bsCmCax
and bsCcPax.

![](https://static.igem.org/mediawiki/2018/4/47/T--Ecuador--N44.2.jpeg)

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.

**September -** Week 45

●
Domestication with overhang PCR of bscA,
bscB and bsCmCax.

![](https://static.igem.org/mediawiki/2018/0/01/T--Ecuador--N45.jpeg)

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.

**September -** Week 46

●
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.

![](https://static.igem.org/mediawiki/2018/4/44/T--Ecuador--N46.jpeg)

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.

**September -** Week 47

●
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

![](https://static.igem.org/mediawiki/2018/6/69/T--Ecuador--N47.jpeg)

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.

**October -** Week 48

●
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

![A group of people sitting in a room  Description generated with very high confidence](https://static.igem.org/mediawiki/2018/0/00/T--Ecuador--N48.jpeg)

**October
-** Week 49

●
Finishing the wiki. **Participation:**
Everyone

![A screenshot of a video game  Description generated with high confidence](https://static.igem.org/mediawiki/2018/7/7d/T--Ecuador--N49.jpeg)

●
Celebration of Birthday of our PI Francisco Flores. Thanks for
everything!!! **Participation:** Everyone.

![](https://static.igem.org/mediawiki/2018/7/75/T--Ecuador--N49.2.jpeg)

**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.

![](https://static.igem.org/mediawiki/2018/e/ea/T--Ecuador--N50.jpeg)

SPONSORS

FOLLOW US

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UNIVERSIDAD DE LAS FUERZAS ARMADAS ESPE.
Ecuador,Sangolquí, Av. General Rumiñahui s/n y Ambato


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Ecuador/Experiments

[iGEM ECUADOR](https://2018.igem.org/Team%3AEcuador)

[PROJECT](https://2018.igem.org/Team%3AEcuador/Description)

[DESCRIPTION](https://2018.igem.org/Team%3AEcuador/Description)
[BACKGROUND](https://2018.igem.org/Team%3AEcuador/Background)
[MODELING](https://2018.igem.org/Team%3AEcuador/Model)
[DESIGN](https://2018.igem.org/Team%3AEcuador/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3AEcuador/Experiments)
[NOTEBOOK](https://2018.igem.org/Team%3AEcuador/Notebook)
[RESULTS](https://2018.igem.org/Team%3AEcuador/Results)

[PARTS](https://2018.igem.org/Team%3AEcuador/Parts)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AEcuador/Public_Engagement)
[BEST INTEGRADED HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Best-In-Human-Pract)

[SAFETY](https://2018.igem.org/Team%3AEcuador/Safety)

[INTERLAB](http://2018.iGEM.org/Team%3AEcuador/Interlab)

[JUDGING FORMS](http://2018.iGEM.org/Team%3AEcuador/Judging)

[TEAM](https://2018.igem.org/Team%3AEcuador/Team)

[TEAM MEMBERS](https://2018.igem.org/Team%3AEcuador/Team)
[COLLABORATIONS](https://2018.igem.org/Team%3AEcuador/Collaborations)

[ATTRIBUTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)

[ATRIBUTTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)
[SPONSORING](https://2018.igem.org/Team%3AEcuador/Sponsoring)

C-lastin, Interlab

# EXPERIMENTS

### Index

[* PHASE I](#P1)

[+ Experiment 1](#P1e1)
[+ Experiment 2](#P1e2)
[+ Experiment 3](#P1e3)
[+ Experiment 4](#P1e4)
[+ Experiment 5](#P1e5)
[+ Experiment 6](#P1e6)
[+ Experiment 7](#P1e7)
[* PHASE II](#P2)

[+ Experiment 1](#P2e1)
[+ Experiment 2](#P2e2)
[+ Experiment 4](#P2e4)
[+ Experiment 5](#P2e5)
[* PHASE III](#P3)

[+ Experiment 1](#P3e1)
[+ Experiment 2](#P3e2)
[+ Experiment 3](#P3e3)
[+ Experiment 4](#P3e4)
[* PHASE IV](#P4)

PHASE I: CONSTRUCTION OF THE COMPOSITE PARTS CBD cipA-BMP2 and CBD cipA-sfGFP

Experiment 1: Competent cells preparation

![](https://static.igem.org/mediawiki/2018/b/b8/T--Ecuador--P1E1.png)

*E.coli* cells DH5α, BL21 (DE3) and BL21 (DE3) pLyss were prepared using the CaCl2 method, slightly modifying the one described in:

[Competent Cells](http://parts.igem.org/Help%3AProtocols/Competent_Cells)

 SHOW/HIDE PROTOCOL

PROTOCOL

1. Autoclave the glass and plastic material (Petri dishes, conical centrifuge tubes, Erlenmeyers) filled ¾ with distilled water to make them free of detergent since any residue is an inhibitor of the growth and transformation of competent cells.
2. Inoculate 125 mL of LB medium with 1 mL of E. coli cell stock and culture overnight at room temperature. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Pre-freeze the conical centrifuge tubes.
4. Transfer the culture to the prechilled conical centrifuge tubes.
5. Centrifuge for 10 minutes at 3000 g at 4 ° C.
6. Gently resuspend by vortexing the cell pellet in 40 mL of ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifuge again for 10 minutes at 3000 g at 4 ° C.
9. Resuspend by pipetting the cell pellet in 15 mL of ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Aliquot in cryotubes 2 mL.
12. Store at -80 ° C.

Preparation of CCMB80 Buffer:

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| CaCl2.2H2O | 11.76 g/L |
| MnCl2.4h2O | 3.95 g/L |
| MgCl2.6H2O | 2.03 g/L |
| KOAc | 0.9815 g/L |
| Glicerol al 10% | 100 mL |

**Note: Adjust the pH to 6.3, sterilize by filtration.**

Experiment 2: Domestication of the gBlocks

![](https://static.igem.org/mediawiki/2018/d/d0/T--Ecuador--P1E2.png)

In order that the restriction enzymes EcoRI and PstI cleavage at the end of the gBlocks and the cohesive ends be generated, a PCR was run using the first forward 5'-ccggaattcgcggccgcttctag-3 'and reverse 5'-aaaactgcagcggccgctactag-3' to add the nucleotide bases to the N-terminal and C-terminal ends.

SHOW/HIDE PROTOCOL

PROTOCOL

The PCR Reaction using Q5 High-Fidelity DNA polymerase was setup according the manufacturer´s instructions (New England Biolabs).

**Table1. PCR Components**

|  |  |  |
| --- | --- | --- |
| **COMPONENT** | **50 uL REACTION** | **FINAL CONCENTRATION** |
| 5X Q5 Buffer | 10 uL | 1X |
| dNTPs (10 mM) | 1 uL | 200 uM |
| Primer Forward (10 uM) | 2.5 uL | 0.5 uM |
| Primer Reverse (10 uM) | 2.5 uL | 0.5 uM |
| Q5 High-Fidelity DNA Polymerase | 0.5 uL | 0.02 U/uL |
| Template DNA (50 ng/uL) | 0.5 uL | 0.5 ng/uL |
| 5X Q5 High GC Enhancer | 10 uL | 1X |
| Nuclease-free water | 23 uL |  |

**Table 2. PCR Conditions**

|  |  |  |
| --- | --- | --- |
| **STEP** | **TEMPERATURE** | **TIME** |
| **Initial denaturation** | 98°C | 30 s |
| **35 ciclos** | 98°C | 10 s |
| 72°C | 25 s |
| 72°C | 25 s |
| **Extensión final** | 72°C | 2 min |
| **Hold** | 4°C |  |

####

Experiment 3: Purification of the PCR products of the gBlocks

The objective of purifying the amplicons of the gBlocks was to use it in the process downstream of assembly 3A. The purification was carried out following the manufacturer's instructions (Invitrogen).

SHOW/HIDE PROTOCOL

Experiment 4 Fusion assembly

![](https://static.igem.org/mediawiki/2018/1/1d/T--Ecuador--P1E4.png)

Once purified products were obtained from the CBD cipA gBlocks (driven by pLac promoter), BMP2 and sfGFP with basepairs added to the N-terminal and C-terminal ends, we proceeded to perform a fusion assembly to obtain the CBD fusion products cipA-BMP2 and CBD cipA-sfGFP. The methodology involved slight variations to the one described in the Open Wetware site [Fusion Assembly](https://openwetware.org/wiki/Prbbbb%3Afusion_assembly_v1).

SHOW/HIDE PROTOCOL

PROTOCOL

1. The purified gBlocks CBD cipA, BMP2 and sfGFP were eluted in Elution Buffer E1 (10 mM Tris-HCl, pH 8.5) while the psb1c3 vector was eluted in Nuclease-free water.
2. Prepare restriction mixes (Table 1) to digest gBlocks

Table 1.

|  |  |  |
| --- | --- | --- |
| **Mix** | **Component** | **2 uL reaction** |
| **A** | 10X NEB Buffer 1.1 | 1.00 uL |
| Nuclease-free water | 0.47 uL |
| EcoRI (15U/uL) | 0.13 uL |
| AgeI (5U/uL) | 0.40 uL |
| **B** | 10X NEB Buffer 4 | 1.00 uL |
| 0.1% BSA | 0.32 uL |
| PstI (10U/uL) | 0.34 uL |
| NgoMIV (10U/uL) | 0.34 uL |
| **C** | 10X Buffer O | 1.16 uL |
| EcoRI (15U/uL) | 0.34 uL |
| PstI (10U/uL) | 0.50 uL |

1. Mix 8.0 uL of purified CBD cipA gBlock (50 ng / uL) with 2 uL of restriction mix A.
2. Mix 8.0 uL of purified BMP2 gBlock (50 ng / uL) with 2 uL of restriction mix B.
3. Mix 8.0 uL of purified gBlock sfGFP (50 ng / uL) with 2 uL of restriction mix B.
4. Mix 3.2 uL of the linearized psb1c3 vector (25 ng / uL) with 0.8 uL of the restriction mix C.
5. Incubate for 2 h at 37 ° C.
6. Inactivate the restriction enzymes heating the samples for 20 min at 85 ° C.9. Prepare the ligation mix (Table 2).

|  |  |
| --- | --- |
| **COMPONENT** | **10 uL REACTION** |
| Nuclease-free water | 7.0 uL |
| 10X T4 DNA Ligase Buffer | 2.0 uL |
| T4 DNA Ligase | 1.0 uL |

1. Mix 4.0 uL CBD cipA digested with 4.0 uL BMP2 digested and 2.0 uL psb1c3 digested vector.
2. Mix 4.0 uL CBD cipA digested with 4.0 uL sfGFP digested and 2.0 uL psb1c3 digested vector.
3. Add 10 uL of the ligation mix to each mix (Step 10 and 11).
4. Incubate overnight at 16 ° C.
5. Inactivate ligase enzyme heating the samples for 10 min at 65 ° C.
6. The volume for transformation depends on the total volume of competent cells (See Transformation protocol).

####

Experiment 5: Transformation

![](https://static.igem.org/mediawiki/2018/1/1d/T--Ecuador--P1E4.png)

The psb1c3-CBD vectors cipA-BMP2 and psb1c3-CBD cipA-sfGFP obtained by fusion assembly were transformed into E. coli DH5α, BL21 (DE3) cells by heath shock method according to the protocol described in [Transformation](http://parts.igem.org/%20Help%3AProtocols/Transformation).

SHOW/HIDE PROTOCOL

PROTOCOL

1. Add 70μl of competent cells in a 2ml Eppendorf tube.
2. Add 8 μl of resuspended DNA in the tube.
Gently pipet up and down several times. Keep all the tubes on ice.3. Close the 2 ml tubes and incubate on ice for 20 minutes
4. For thermal shock place the tubes in the water bath at 42 ° C for 45 seconds
The 2 ml tubes should be in a rack for floating foam tubes. Place in a water bath to ensure that the bottom of the tubes is submerged. Time is critical5. Incubate on ice for 2-4 minutes
6. Add 800μl of SOC medium in each transformation (Table 1)

 **Table 1. SOC medium composition**

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Triptone | 10g/L |
| Yeast extract | 5g/L |
| NaCl | 0,5g/L |
| MgSO 4 | 10mM |
| Glucose | 20 mM Glucose \* (thermosensitive)    + Adjust to pH 7 and add these compounds after sterilizing in autoclave |

7. Incubate for 1.3 h at 37 ° C.
8. Centrifuge the tubes for 3 minutes at 7000 g.
9. Discard 400μL of the supernatant. Resuspend cells in the remaining 100 μL.
10. Spread by extension 100 μL of the transformed product in Petri dishes containing
    LB + chloramphenicol medium (Table 2).

 **Table 2. Agar LB+cloranfenicol**

|  |  |
| --- | --- |
| **Component** | **Concentration** |
| Triptone | 10g/L |
| Yeast extract | 5g/L |
| NaCl | 0,5g/L |
| Agar | 15g/L |
| Chloramphenicol | 1 mL of a solution of Chloramphenicol at 25 mg / mL \* (thermosensitive)    + Add this compound after sterilizing in autoclave |

11. Incubate overnight transformations at 37 ° C.

####

Experiment 6: Extraction of plasmid DNA

![](https://static.igem.org/mediawiki/2018/f/f8/T--Ecuador--P1E6.png)

Plasmid DNA was extracted from the bacterial transformation products for subsequent enzymatic digestions and PCR reactions to verify that the psb1c3-CBD inserts cipA-BMP2 and psb1c3-CBD cipA-sfGFP are correct.

SHOW/HIDE PROTOCOL

PROTOCOL

* Add 1.5 mL of bacterial culture in a 2 mL tube.
* Centrifuge at 13,000 rpm for 45 seconds at 4 ° C.
* Resuspend the pellet in 250 μL cold lysis solution 1 (with RNAase), homogenize vortex.

**Table1. Lysis solution preparation**

|  |  |  |
| --- | --- | --- |
| **Lysis solution 1** | **Lysis solution 2** | **Lysis solution 3** |
| 50mM de glucosa   25mM de Tris-HCl pH 8   10mM EDTA pH 8   RNasa 1 ug/mL | 0.2 N de NaOH  1% (w/v) de SDS | 5M of potassium acetate dissolve with glacial acetic acid and gauge with distilled water |

* Incubate 2 minutes at room temperature.Add 250 μL of lysis 2, homogenize by inversion.
* Incubate 5 minutes on ice.
* Add 300 μL cold lysis solution 3, homogenize by inversión
* Incubate on ice for 10 minutes.
* Centrifuge at 14,000 rpm for 10 minutes at 4 ° C.
* Transfer supernatant and add two volumes of absolute cold ethanol or absolute isopropanol, homogenize by inversion.
* Incubate at -20 ° C for 20 minutes.l. Centrifuge 14000 rpm for 30 minutes.m. Discard the supernatant.n. Add 1 mL of 70% ethanol and mix by inversion.or.
* Centrifuge at 14,000 rpm for 20 minutes.
* Discard the supernatant and let it dry for 15 minutes.
* Resuspend in 25 μL of water DPC.

**Table 2. Materials for Plasmidic extraction**

|  |  |
| --- | --- |
| **Biological / equipment** | **Indications** |
| **bacterial culture** | 3 mL of 24 hour liquid bacterial culture |
| **2ml microtubes** | Label the tubes with the name of the part or location of the well before beginning |
| **Ice Cube** | Fill the bucket with ice and pre-cool the 2mL tubes (5 minutes). Thaw stocks of competent ice cubes (10-15min) |
| **Timer** | Preferably with temperature control |
| **2ml tube centrifuge** | nuclease-free water |

####

Experiment 7: Enzymatic digestion of the plasmids containing PSB1C3 as backbone

![](https://static.igem.org/mediawiki/2018/4/43/T--Ecuador--P1E7.png)

The psb1c3-CBD plasmid products cipA-BMP2 and psb1c3-CBD cipA-sfGFP were digested with EcoRI and PstI to distinguish the size of the products by means of a 1% agarose gel electrophoresis.

SHOW/HIDE PROTOCOL

PROTOCOL

1. Prepare the EcoRI-PstI restriction mix.

|  |  |
| --- | --- |
| **COMPONENT** | **3 uL reaction** |
| 10X Buffer O | 1.74 uL |
| EcoRI (15U/uL) | 0.51 uL |
| PstI (10U/uL) | 0.75 uL |

1. Mix 3 uL of the restriction mix EcoRI-PstI with 6 uL of the extracted plasmid.
2. Incubate for 2 h at 37 ° C.
3. Heat inactivation for 20 min at 85 ° C.

PHASE II: CONSTRUCTON OF THE COMPOSITE PART CBD cipA-ELP

Experiment 1: Amplification of ELP from pET-24a-ELP[V-150]

A PCR reaction was assessed to try amplifying the ELP[V-150] (transition temperature=28.6°C) from the commercial plasmid pET-24a-ELP[V-150] (Addgene). Numerous attempts were done using a set of primers; however, similar electrophoresis patterns with multiple diffuse bands were obtained.

[Competent Cells](http://parts.igem.org/Help%3AProtocols/Competent_Cells)

SHOW/HIDE PROTOCOL

**Protocol of the PCRs tested**
The PCR Reaction using Dream Taq DNA polymerase was setup according the manufacturer´s instructions (Thermo Scientific).
**Table1. PCR Components**

|  |  |  |
| --- | --- | --- |
| **COMPONENT** | **50 uL REACTION** | **FINAL CONCENTRATION** |
| DreamTaq PCR Master Mix (2X) | 25 uL | 1X |
| Primer Forward (10 uM) | 2.5 uL | 0.5 uM |
| Primer Reverse (10 uM) | 2.5 uL | 0.5 uM |
| Template DNA (10 ng/uL) | 3.0 uL | 0.6 ng/uL |
| Nuclease-free water | 17 uL |  |

**Table 2. PCR Conditions**

|  |  |  |
| --- | --- | --- |
| **STEP** | **TEMPERATURE** | **TIME** |
| **Initial denaturation** | 95°C | 3 min |
| **Round 1**   **(5 cycles)** | 95°C | 30 s |
| T annealing Round 1\* | 30 s |
| 72°C | 1.4 min |
| **Round 2**   **(25 cycles)** | 95°C | 30 s |
| T annealing Round 2\* | 30 s |
| 72°C | 1.4 min |
| **Hold** | 4°C |  |

**Table 3.** **Primers used in the PCRs tested**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **PCR** | **Primers** | **Primer sequence** | **T annealing Round 1** | **T annealing Round 2** |
| **1** | Forward 1 | 5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3' | 44°C | 65.5°C |
| Reverse 1 | 5'-ctgcagcggccgctactagtattaaccggtccagggaactccaac-3' |
| **2** | Forward 1 | 5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3' | 45.5°C | 67°C |
| Reverse 2 | 5'-ctgcagcggccgctactagtattaaccggtcggccagggaactcc-3' |
| **3** | Forward 1 | 5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3' | 44°C | 65.5°C |
| Reverse 3 | 5'-ctgcagcggccgctactagtattaaccggtcggccagggaact-3' |
| **4** | Forward 2 | 5'-agaaggaggagtacatatg-3' | 42.5°C | |
| Reverse 4 | 5'-gatcctgaagattatcacgg-3' |

**Purification protocol of DNA** **fragment from agarose gel**
**Please refer to:**
<https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink_gel_extraction_pcr_combo_man.pdf>

####

Experiment 2: Domestication of the ELP550 amplicon

OOnce primers was purified, the ELP550 obtained with the Forward 1 (5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3 ') and Reverse 2 (5'-ctgcagcggccgctactagtattaaccggtcggccagggaactcc-3') a PCR was run using the ForwardFu primers (5'-ccggaattcgcggccgcttctag-3 ') and ReverseFu (5'-aaaactgcagcggccgctactag-3') to add the nucleotide bases 5'-ccg-3 'and 5'-aaaac-3' to the N-terminus and C-terminus, respectively, and consequently leave space so that the enzymes EcoRI and PstI cut near the ends.
The PCR product was purified using the Quick Gel Extraction and PCR Purification Combo Kit according to the manufacturer's instructions (Invitrogen).

SHOW/HIDE PROTOCOL

**PCR SHOW/HIDE PROTOCOL**
The PCR Reaction using Dream Taq DNA polymerase was setup according the manufacturer´s instructions (Thermo Scientific).
**Table1. PCR Components**

|  |  |  |
| --- | --- | --- |
| **COMPONENT** | **50 uL REACTION** | **FINAL CONCENTRATION** |
| DreamTaq PCR Master Mix (2X) | 25 uL | 1X |
| Primer Forward (10 uM) | 2.5 uL | 0.5 uM |
| Primer Reverse (10 uM) | 2.5 uL | 0.5 uM |
| Template DNA (60 ng/uL) | 1.0 uL | 1.2 ng/uL |
| Nuclease-free water | 19 uL |  |

**Table 2. PCR Conditions**

|  |  |  |
| --- | --- | --- |
| **STEP** | **TEMPERATURE** | **TIME** |
| **Initial denaturation** | 95°C | 3 min |
| **Round 1**   **(5 cycles)** | 95°C | 30 s |
| 54°C | 30 s |
| 72°C | 30 s |
| **Round 2**   **(25 cycles)** | 95°C | 30 s |
| 57°C | 30 s |
| 72°C | 30 s |
| **Hold** | 4°C |  |

####

Experiment 4 Fusion assembly

![](https://static.igem.org/mediawiki/2018/1/1d/T--Ecuador--P1E4.png)

Once purified products were obtained from the CBD cipA gBlocks (driven by pLac promoter), BMP2 and sfGFP with basepairs added to the N-terminal and C-terminal ends, we proceeded to perform a fusion assembly to obtain the CBD fusion products cipA-BMP2 and CBD cipA-sfGFP. The methodology involved slight variations to the one described in the Open Wetware site [Fusion Assembly](https://openwetware.org/wiki/Prbbbb%3Afusion_assembly_v1).

#### SHOW/HIDE PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Experiment 5: Transformation

![](https://static.igem.org/mediawiki/2018/1/1d/T--Ecuador--P1E4.png)

See Transformation Protocol, Phase I.

#### SHOW/HIDE PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Phase III: Construction of the cellulose production module

Experiment 1: Amplification of LacI from pET-24a-ELP[V-150]

![](https://static.igem.org/mediawiki/2018/3/3b/T--Ecuador--P3E1F1.png)

![](https://static.igem.org/mediawiki/2018/3/35/T--Ecuador--P3E1F2.png)

Due to the need of an induce mechanism we decided to use LacI cassette in our assemblies. To obtain this cassette we did a PCR reaction to amplify the LacI (promoter and ORF) from the commercial plasmid pET-24a-ELP[V-150] (Addgene).

#### SHOW/HIDE PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Experiment 2: Domestication of the gblocks and the LacI amplicon

![](https://static.igem.org/mediawiki/2018/1/18/T--Ecuador--P3E2.png)

In order to obtain specific sequences ends to be digested by the gibson assembly exonuclease, we used specific primers for each sequence to add overhanging ends to the sequence that must be ligated. This overhanging ends were added by PCR with specific conditions for each sequence.

#### SHOW/HIDE PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Experiment 3: Purification of the PCR products of the gBlocks

The objective of purifying the amplicons of the gBlocks was to use it in the process downstream of assembly 3A. The purification was carried out following the manufacturer's instructions (Invitrogen).

**Experiment 3: Linealization of the psb1C3 and psb1A3 plasmids.**

![](https://static.igem.org/mediawiki/2018/d/d3/T--Ecuador--P3E3F1.png)
![](https://static.igem.org/mediawiki/2018/c/c8/T--Ecuador--P3E3F2.png)

To assemble all the parts to the plasmids with Gibson technique, we made the linearization and removal of the biobrick suffix and prefix from the psb1C3 and psb1A3 through PCR.

#### SHOW/HIDE PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Experiment 4: Gibson assembly

![](https://static.igem.org/mediawiki/2018/d/d6/T--Ecuador--P3E4.png)

Several trials were carried out to assemble two vectors with the cellulose production module as planned. We faced with several problems that occurred during this trial and we could not obtain the assembled vectors that we wanted, but we obtained degraded DNA seen in electrophoresis and this could be due to the high concentration of GC at the ends of the sequences that interfer with the Gibson’s exonuclease cleavage activity.

Phase IV: Characterization of the binding strength of the CBD cipA with an endogenous C-terminal linker

Measurements of remaining fluorescences, after different washes, were done according to the protocol described by the Team Imperial 2014. Briefly, 20g of kombucha cellulose were blended with 125 mL. Then, 100 uL of the mixture were added into each well of a 96-plate reader and dried at 60º for 5.5 hours using a stove. A volume of 100 uL of CBD cipA-sfGFP protein extract was added into each well an incubated at 4ºC overnight. The fluorescence was measured after washing with different substances: dH20, 70% EtOH, Plasma and Stif.

SPONSORS

FOLLOW US

CONTACT US

biosin.espe@outlook.es

UNIVERSIDAD DE LAS FUERZAS ARMADAS ESPE.
Ecuador,Sangolquí, Av. General Rumiñahui s/n y Ambato

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Ecuador/Results

[iGEM ECUADOR](https://2018.igem.org/Team%3AEcuador)

[PROJECT](https://2018.igem.org/Team%3AEcuador/Description)

[DESCRIPTION](https://2018.igem.org/Team%3AEcuador/Description)
[BACKGROUND](https://2018.igem.org/Team%3AEcuador/Background)
[MODELING](https://2018.igem.org/Team%3AEcuador/Model)
[DESIGN](https://2018.igem.org/Team%3AEcuador/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3AEcuador/Experiments)
[NOTEBOOK](https://2018.igem.org/Team%3AEcuador/Notebook)
[RESULTS](https://2018.igem.org/Team%3AEcuador/Results)

[PARTS](https://2018.igem.org/Team%3AEcuador/Parts)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)

[HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Human_Practices)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AEcuador/Public_Engagement)
[BEST INTEGRADED HUMAN PRACTICES](https://2018.igem.org/Team%3AEcuador/Best-In-Human-Pract)

[SAFETY](https://2018.igem.org/Team%3AEcuador/Safety)

[INTERLAB](http://2018.iGEM.org/Team%3AEcuador/Interlab)

[JUDGING FORMS](http://2018.iGEM.org/Team%3AEcuador/Judging)

[TEAM](https://2018.igem.org/Team%3AEcuador/Team)

[TEAM MEMBERS](https://2018.igem.org/Team%3AEcuador/Team)
[COLLABORATIONS](https://2018.igem.org/Team%3AEcuador/Collaborations)

[ATTRIBUTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)

[ATRIBUTTIONS](https://2018.igem.org/Team%3AEcuador/Attributions)
[SPONSORING](https://2018.igem.org/Team%3AEcuador/Sponsoring)

`

C-lastin, Interlab

# RESULTS

### Index

[* PHASE I](#P1)

[+ Experiment 3](#l1)
[+ Experiment 5](#l2)
[+ Experiment 6](#l3)
[+ Experiment 7](#l4)
[* PHASE II](#P2)

[+ Experiment 1](#l5)
[+ Experiment 2](#l6)
[* PHASE III](#P3)

[+ Experiment 1](#l7)

PHASE I: CONSTRUCTION OF THE COMPOSITE PARTS CBD cipA-BMP2 and CBD cipA-sfGFP

**Experiment 3: Purification of the PCR products of the gBlocks**

![](https://static.igem.org/mediawiki/2018/1/1f/T--Ecuador--RE3.png)

Lane 1: 1kb DNA ladder
Lane 6: Purified PCR product of CBD gBlock
Lane 7: Purified PCR product of BMP2 gBlock
Lane 8: Purified PCR product of sfGFP gBlock
**Gel composition**
Agarose gel 1%
1X TAE Buffer

[Competent Cells](http://parts.igem.org/Help%3AProtocols/Competent_Cells)

Experiment 5: Transformation protocol

![](https://static.igem.org/mediawiki/2018/e/e9/T--Ecuador--RE5.png)

EXPERIMENT 6: Extraction of plasmid DNA

![](https://static.igem.org/mediawiki/2018/7/75/T--Ecuador--RE7.png)

Agarose gel 1%
1X TAE Buffer
Lane 1: High Mass DNA ladder
Lane 2: psb1c3-CBD cipA-sfGFP plasmid
Lane 3: psb1c3-CBD cipA-BMP2 plasmid

EXPERIMENT 7: Enzymatic digestion of the plasmids containing psb1c3 as backbone

![](https://static.igem.org/mediawiki/2018/5/5f/T--Ecuador--REE7.png)

Agarose gel 1%
1X TAE Buffer
Lane 1: High Mass DNA Ladder
Lane 2-4: Digested product of psb1c3-CBD cipA-BMP2.
Lane 5: Digested product of psb1c3-CBD cipA-sfGFP.

PHASE II: CONSTRUCTON OF THE COMPOSITE PART CBD cipA-ELP

Experiment 1: Amplification of ELP from pET-24a-ELP[V-150]

![](https://static.igem.org/mediawiki/2018/b/bc/T--Ecuador--RP2E1.png)

Agarose gel 1%
1X TAE Buffer
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

Experiment 2: Domestication of the ELP550 amplicon

![](https://static.igem.org/mediawiki/2018/e/e0/T--Ecuador--RP2E2.png)

Agarose gel 1%
1X TAE Buffer
Lane 1: 100 pb DNA ladder
Lane 2: Purified ELP550
Lane 3: No sample
Lane 3: Unpurified ELP550

#### PROTOCOLO

1. Autoclavar el material de vidrio y plástico (cajas Petri, conical centrifuge tubes, erlenmeyers) filled ¾ con agua destilada para hacerlos libre de detergente ya que cualquier residuo es un inhibidor del crecimiento y transformación de células competentes.
2. Inocular 125 mL de medio LB con 1 mL de seed stock de células E. coli y cultivarlas overnight a temperatura ambiente. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
3. Precongelar los conical centrifuge tubes.
4. Transferir el cultivo a los prechilled conical centrifuge tubes.
5. Centrifugar por 10 minutos en 3000 g a 4°C.
6. Resuspender gentilmente by vortexing el pellet celular en 40 mL de ice cold CCMB80 buffer.
7. Hold on ice for 20 minutes.
8. Centrifugar nuevamente por 10 minutos en 3000 g a 4°C.
9. Resuspender by pipetting el pellet celular en 15 mL de ice cold CCMB80 buffer.
10. Hold on ice for 20 minutes.
11. Alicuotar en criotubes 2 mL.
12. Almacenar a -80°C.
CCMB80 Buffer

|  |  |
| --- | --- |
| Componente | Concentración |
| **CaCl2.2H2O** | 11.76 g/L |
| **MnCl2.4h2O** | 3.95 g/L |
| **MgCl2.6H2O** | 2.03 g/L |
| **KOAc** | 0.9815 g/L |
| **Glicerol al 10%** | 100 mL |

pH 6.3.
Esterilizar por filtración

Phase III: Construction of the cellulose production module

Experiment 1: Amplification of LacI from pET-24a-ELP[V-150]

![](https://static.igem.org/mediawiki/2018/9/9e/T--Ecuador--R3F1.png)

Agarose gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder

Lane B: BscA 2536 bp
Lane C: BscB 2827 bp
Lane D: empty
Lane E: BscC2 2139 bp

![](https://static.igem.org/mediawiki/2018/6/61/T--Ecuador--R3F2.png)

Agarose gel at 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder
Lane B: bscC1 2152 bp

![](https://static.igem.org/mediawiki/2018/9/9e/T--Ecuador--R3F1.png)

Agarose gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder

Lane B: BscA 2536 bp
Lane C: BscB 2827 bp
Lane D: empty
Lane E: BscC2 2139 bp

![](https://static.igem.org/mediawiki/2018/1/1a/T--Ecuador--R3F3.png)

Agarose gel at 1%
1X TAE Buffer
Lane A: 100 pb DNA ladder
Lane B: unidentified sample
Lane C: BscD 849 pb
Lane D: BscD 849 pb

The parts to be assembled with psb1C3 were amplified using 8% DMSO due to its primers with high concentration of GC and the tendency to form secondary structures. As shown in Figures 1, 2 and 3, all the amplicons were of the expected length. The BscD was the only sequence that after the PCR still had a nonspecific amplicon, but its fluorescence was weaker than the fluorescence of the expected length band (figure 3).

![](https://static.igem.org/mediawiki/2018/9/98/T--Ecuador--R3F4.png)

Agarose gel at 1%
1X TAE Buffer
Lane A: 100 pb DNA ladder
Lane B: lineal Psb1C3 2135 bp
Lane C: lineal Psb1A3 2050 bp
Lane D: bsCmcax 1522 bp
Lane E:bsCcpax (1st attempt) 1512 bp
Lane F:bsCcpax (2nd attempt) 1512 bp
Lane G: bsCcpax (3st attempt) 1512 bp
Lane H: LacI 1420 bp

The parts to be assembled with psb1A3 were also amplified using 8% DMSO for the same reason explained above. As shown in Figure 4 all the amplicons were of the expected length. The BsCcpax PCR attempts gave us different quality amplicons using different annealing temperatures.

![](https://static.igem.org/mediawiki/2018/7/74/T--Ecuador--R3F5.png)

Agarose Gel 1%
1X TAE Buffer
Lane A: 1 kb DNA ladder
Lane B: gibson assembly psb1C3
Lane C: gibson assembly psb1A3

Using the Gibson assembly kit instructions from NEB we could not see any bands representing the expected ligation product for both plasmids (psb1C3 and psb1A3) (figure 5). A later attempt was done using DMSO, but no change was seen.

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biosin.espe@outlook.es

UNIVERSIDAD DE LAS FUERZAS ARMADAS ESPE.
Ecuador,Sangolquí, Av. General Rumiñahui s/n y Ambato