iGEM 2017 — Team UChile_OpenBio-CeBiB

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# Team:UChile OpenBio-CeBiB/Notebook

Document

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* [Project](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)

  [Description](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)
  [Parts](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Parts)
  [Procedures](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Experiments)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Results)
  [Attributions](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Attributions)
* [HP](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)

  [HP Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)
  [Collaborations](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Collaborations)
  [Silver](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Silver)
  [Gold](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Gold_Integrated)
  [Engagement](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Engagement)
* [Modelling](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)

  [Modelling Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)
  [Procedure](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Procedure)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Results)
* [Safety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety)

  [Biosafety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/Biosafety)
  [Lab Protocols](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/LabProtocols)
* [Notebook(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Notebook)
* [Team(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Team)
* [Giant Jamboree](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree)

  [Our experience](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/OurExperience)
  [Judging Feedback](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/JudgingFeedback)
  [Photos](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/Photos)
* [Home(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB)

# Notebook

Week 1 [10/17/16]

Presentation of 5 projects in the Engineering and Science Festival.
 Workshops are made daily for high school students and families, aprox 300 peoples were in our Workshops!!

Week 2 [10/24/16]

Presentation of the projects to the academic body of the Chemical and Biotechnological Enginnering Department.

Week 3 [10/31/16]

Project selection, Greenhardtii was elected above the other 4 projects

Week 4 [11/07/16]

First work meeting, selecting an appropiate organism capable of doing photosynthesis.
First presentation of the project to public.

Week 5 [11/14/16]

Discussion of a possible production of a biomaterial, team is divided on 3 commisions to search for biomaterials.

Week 6 [11/21/16]

The team find out fatty acids, cellulose and natural inks. The commisions previously formed investigate possible production of each biomaterial.

Week 7 [11/28/16]

Presentation of the methabolics routes in the production of the biomaterials, cellulose is elected as the most potentialy produced biomaterial.

Week 8 [12/05/16]

No lab activity this week, University Final Exams.

Week 9 [12/12/16]

No lab activity this week, University Final Exams.

Week 10 [12/18/16]

Meeting to form new commisions for working during holidays, Cellulose production and CO2 capture optimization.

Week 11 [12/26/16]

No activity in lab this week, University Exams.

Week 12 [01/02/17]

Holidays!!!

Week 13 [01/09/17]

Cellulose comission investigate bacterial cellulose properties.

Week 14 [01/16/17]

Cellulose comission arrange a pizza meeting with José Duguet, so we could develop a regulation system for production.
Sofi has joined the party

Week 15 [01/23/17]

Cellulose comission finds the inducible promoters and investigate them.

Week 16 [01/30/17]

Presentation of the research done during January.

Week 17 [02/06/17]

Holly Holidays!!

Week 18 [02/13/17]

Holly Holidays!!

Week 19 [02/20/17]

Holly Holidays!!

Week 20 [02/27/17]

Holly Holidays!!

Week 21 [03/06/17]

Clinging a little bit longer to summer vacations.

Week 22 [03/13/17]

Actualization meeting after holidays, we start to search for financial support.

Week 23 [03/20/17]

Presentation to CeBiB asking for financial and academic support.
Stay selling in the University for friday parties.

Week 24 [03/27/17]

Attending to the MIT Global Start-Up Workshop, for learning how to do a correct elevator pitch.

Week 25 [04/03/17]

Meeting with professor Franck Quero, we chat about types of cellulose and more of its wordeful properties

Week 26 [04/10/17]

Working on Franck Quero's lab, growing some *G. xilinum*.

Week 27 [04/17/17]

Collecting our bacterial cellulose!!!

Week 28 [04/24/17]

No activity in lab this week

Week 29 [05/01/17]

No activity in lab this week

Week 30 [05/08/17]

No activity in lab this week

Week 31 [05/15/17]

No activity in lab this week

Week 32 [05/22/17]

Lauching our online Survey

Week 33 [05/29/17]

Meeting with the team in order to determinate the list of protocols that we were going to get for the later lab work.

Week 34 [06/05/17]

No activity in lab this week

Week 35 [06/12/17]

It is possible to elaborate a first document with the experimental strategy and most of the protocols. It was presented to professor Álvaro, who suggested us to correct some details and contact Dr. Tomás egaña, Engineer in Molecular Biotechnology, expert working with the microalgae *Chlamydomonas reinhardtii*.

Reunion with the team to find the answers of the doubts about the document with the elaborated protocols.

Participation on the radio show "I want to be scientific" ("Quiero ser científico") with high school students.
Exposition of the project to kids in the science fair held at Ñuñoa square.

Week 36 [06/19/17]

Meeting with Dr. Tomás Egaña, who kindly helped us solve our doubts about our experimental strategy and some protocols related to *Chlamydomonas reinhardtii*. Finally, he talked us about Myra Chávez, a postdoc and an expert working with *Chlamydomonas reinhardtii*, who could share with us transformation plasmids, and other materials.

Discussion about the answers made in the las meeting and the mathematical model

Week 37 [06/26/17]

We achieved to make contact with Myra Chávez, who offered us help to solve some problems and she kindly gave us pBC1-CrGFP. From now on, Myra became a big help and support for our team and we nicknamed her as Maymi.

Week 38 [07/03/17]

Modellating meeting, looking for the parameters from Brenda

We launched our crowdfunding video! [you can watch it if you like](https://www.youtube.com/watch?v=g0HHWjY87GM)

Our first and longer collaboration started with iGEM Pasteur

Week 39 [07/10/17]

Meeting to work in the sequences that must be sent to print, define, and analyze. We also searched the tools of optimization of codons for *Chlamydomonas reinhardtii* to make the first order to IDT.

Meeting with Lucho :D , Some modellating problems are solved and was proposed for the next meeting, have the most efficient promoter

Week 40 [07/17/17]

We improved the genetic circuit. Maymi accepted to be part of the team.

Find out that it is almost impossible to synthesize bacterial cellulose, K.O. cellulose production :(

Week 41 [07/24/17]

Usage of the IDT tool to make the codon’s optimization.

Week 42 [07/31/17]

Collaboration from Germany: LMU München (Ludwig Maximilians Universität München), they helped us check our experimental strategy and sequences design.

Week 43 [08/07/17]

Meeting with Jose Duguet, who give us information about how to make the iGEM parts and helped us with some questions. We then worked in the parts design.

Week 44 [08/14/17]

Meeting with professor Álvaro, design of the primers, and consultations.

Week 45 [08/21/17]

Collaboration from England, Dr. Allison Smith from the Cambridge University. She shared us the promoter MetE sequence, 5’UTR and its terminator. She also gave us some advices for the elaboration of our construct.

Finished the primers design.

The sequences were finally ready to be sent to IDT.

The shipment of the sequences was made to IDT.

Week 46 [08/28/17]

Meeting with Maymi and professor Álvaro to specify the experimental strategy, materials and required methods.

Week 47 [09/04/17]

Amplification of pBC1-CrGFP in  *E. coli*  DH10B chemically competent.

Repetition of the last lab. Because there was no growth on the plate, the amplification was repeated. However, now the transformation was made in the *E. coli* top 10 chemically competent strains with more care and caution. After a thermic shock and incubation TB medium for an hour and a half, the cells were transfered to several plates with LB and ampicillin, and they were left incubating at 37°C.

The plates were checked and there actually was presence of transformant colonies with resistance to ampicillin, two of them were inoculated in Falcon tubes with LB and ampicillin medium at 37°C.

Miniprep of the transformants colonies in Falcon. Isolation of the plasmid pBC1-CrGFP.

First aproach to design our poster!!!

Week 48 [09/11/17]

Test transformation of the *Chlamydomonas reinhardtii* by method of glass beads with pBC1-CrGFP. We also learnt to cell count on a Neubauer camera. After the transformation the cells were left incubating in Falcon tubes with TAPS medium in dark during the night at room temperature with agitation.

Continuing with the last lab. The cells were incubated in TAPS, then they were centrifuged and sowed in plates with TAPS medium with agar and paromomycin. They were left incubating at ambient temperature.

Week 49 [09/18/17]

Meeting in the Energy Ministry

Week 50 [09/25/17]

Due to that we were informed that some of the sequences that were sent could not be printed, a second order was made to IDT with the correct sequences.

Week 51 [10/02/17]

Arrival of the first order from IDT.

Resuspension of the lyophilized gBlocks.

It was informed to us that one of the sequences from the second order could not be synthesized again.

Workshop on the XI Science and Technology Fair in the Metropolitan park, we have kids visiting us!!!

Week 52 [10/09/17]

Resuspension of the universal primers and PCR of the gBlock of the first order from IDT.

Preparation of gel extraction of amplified sequences cut DNA bands.

Visit to Los Maitenes and Focus Group with the neighbors

Chemical and Biotechnological Week were we explained what is a bioreactor and lauch a contest.

Week 53 [10/16/17]

Extraction and purification of DNA from the agarose gel bands.

Digestion of the standard sequences and the igem vector pSB1C3. Ligation with the standard sequences.

Transformation by electroporation of the vectors and sequences in BL21DE3 E. coli strain. Seeding in selective mediums, they were left incubating at 37°C until 2 p.m. of the next day.

Isolation of the colonies and growth in LB liquid medium with antibiotic. Left at 4°C

Attendance to the VI National Congress of Biotechnology held in Concepción

Week 54 [10/23/17]

Miniprep of the colonies and generation of a glycerol inventory.

First test of miniprep digestion. The samples were saved at -20°C.

Preparation of visualization gel of the miniprep digestion. Bands on the gel were not observed.

The visualization gel was repeated and again bands on the gel were not observed. The part was sent.

We decided to repeat the test of miniprep digestion and electrophoresis was carried out. Finally, we were able to observe the bands, which validated the construct with the part that was sent.

Week 55 [10/30/17]

VERY HARD WORK in wiki


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# Team:UChile OpenBio-CeBiB/Experiments

Document

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* [Project](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)

  [Description](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)
  [Parts](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Parts)
  [Procedures](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Experiments)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Results)
  [Attributions](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Attributions)
* [HP](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)

  [HP Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)
  [Collaborations](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Collaborations)
  [Silver](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Silver)
  [Gold](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Gold_Integrated)
  [Engagement](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Engagement)
* [Modelling](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)

  [Modelling Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)
  [Procedure](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Procedure)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Results)
* [Safety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety)

  [Biosafety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/Biosafety)
  [Lab Protocols](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/LabProtocols)
* [Notebook(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Notebook)
* [Team(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Team)
* [Giant Jamboree](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree)

  [Our experience](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/OurExperience)
  [Judging Feedback](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/JudgingFeedback)
  [Photos](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/Photos)
* [Home(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB)

# Procedure

The main objetives of this project, at first instance, is to develope a Chlamydomonas reinhardtii strain that is able to fix more quantities of CO2 and, at second instance, once got the ability, produce recombinant proteins that the other strains are not capable to do. On the other hand, the second aim is to characterized kineticallyan adjustable promoter for vitamin B12, which was nicely shared by Dr. Alisson Smith.

That is why, our team has been working on an experimental method that is explained in the next figure:

Figure I: Scheme of our plan, which the wall deficient cells are UVM4 and UVM11 strain

[![](https://static.igem.org/mediawiki/2017/8/84/T--UChile_OpenBio-CeBiB--LogoCebib.png)
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# Team:UChile OpenBio-CeBiB/Results

Document

[![](https://static.igem.org/mediawiki/2017/9/9d/T--UChile_OpenBio-CeBiB--logo1.png)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB)

* [Project](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)

  [Description](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Description)
  [Parts](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Parts)
  [Procedures](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Experiments)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Results)
  [Attributions](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Attributions)
* [HP](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)

  [HP Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP)
  [Collaborations](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Collaborations)
  [Silver](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Silver)
  [Gold](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/HP/Gold_Integrated)
  [Engagement](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Engagement)
* [Modelling](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)

  [Modelling Overview](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model)
  [Procedure](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Procedure)
  [Results](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Model/Results)
* [Safety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety)

  [Biosafety](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/Biosafety)
  [Lab Protocols](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Safety/LabProtocols)
* [Notebook(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Notebook)
* [Team(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Team)
* [Giant Jamboree](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree)

  [Our experience](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/OurExperience)
  [Judging Feedback](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/JudgingFeedback)
  [Photos](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB/Jamboree/Photos)
* [Home(current)](https://2017.igem.org/Team%3AUChile_OpenBio-CeBiB)

# Results

Here are the lab results

**First amplification of pBC1. There was no growth of chemicompetent transformants [09/05/2017].**

![](https://static.igem.org/mediawiki/2017/f/fb/T--UChile_OpenBio-CeBiB--results1.png)

**pBC1 Amplification. Thermal shock transformation [09/06/2017].**

![](https://static.igem.org/mediawiki/2017/f/f4/T--UChile_OpenBio-CeBiB--results2.png)

![](https://static.igem.org/mediawiki/2017/f/fe/T--UChile_OpenBio-CeBiB--results3.png )

![](https://static.igem.org/mediawiki/2017/b/b8/T--UChile_OpenBio-CeBiB--results4.png )

**Electrophoresis results [10/13/2017]**

![](https://static.igem.org/mediawiki/2017/2/2b/T--UChile_OpenBio-CeBiB--results5.png )

Gel visualization of amplification of the gblocks Est2 and Est4

![](https://static.igem.org/mediawiki/2017/1/12/T--UChile_OpenBio-CeBiB--results6.png )

Gel visualization of the sequences of the second order made to IDT

![](https://static.igem.org/mediawiki/2017/8/84/T--UChile_OpenBio-CeBiB--results7.png )

Gel extraction for purification of the gBlock of the second order made to IDT

**Selected colonies for miniprep [10/19/17]**

![](https://static.igem.org/mediawiki/2017/9/91/T--UChile_OpenBio-CeBiB--results8.png )

LB and ampicillin 100 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pBC1-Se 1\_2. It grows.

![](https://static.igem.org/mediawiki/2017/9/99/T--UChile_OpenBio-CeBiB--results9.png )

LB and ampicillin 100 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pBC1-Se 2\_2. It grows.

![](https://static.igem.org/mediawiki/2017/b/b7/T--UChile_OpenBio-CeBiB--results10.png )

LB and ampicillin 100 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pBS1-SEQ. It do not grows.

![](https://static.igem.org/mediawiki/2017/d/d9/T--UChile_OpenBio-CeBiB--results11.png )

LB and Chloranfenicol 50 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pSB1C3-Est1\_2. It grows.

![](https://static.igem.org/mediawiki/2017/7/77/T--UChile_OpenBio-CeBiB--results12.png )

LB and Chloranfenicol 50 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pSB1C3-Est2. It grows

![]( https://static.igem.org/mediawiki/2017/9/96/T--UChile_OpenBio-CeBiB--results13.png)

LB and Chloranfenicol 50 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pSB1C3. It grows

![](https://static.igem.org/mediawiki/2017/3/3f/T--UChile_OpenBio-CeBiB--results14.png )

LB and Ampicillin 100 ug/ml. BL21DE3 strain of E. coli (No TOP10E) transformed by pBC1. It grows

**Results of the third restriction test**

![]( https://static.igem.org/mediawiki/2017/b/b4/T--UChile_OpenBio-CeBiB--results15.png)

Superior gel. It is indicated with (N °) the colony from which the plasmid comes. Staircase of 1 kB. Nomenclature: Part-N ° of colony-Plasmid to which the part is bound (Lig plasmid)

![](https://static.igem.org/mediawiki/2017/b/be/T--UChile_OpenBio-CeBiB--results16.png )

Lower gel. It is indicated with (N °) the colony from which the plasmid comes. Staircase of 1 kB. Nomenclature: Part-N ° of colony-Plasmid to which the part is bound (Lig plasmid).

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