Team:UC Chile2/SpiderColi/Notepad

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Revision as of 01:13, 16 August 2012

March, 5 - 11, 2012

The whole team met for the first time after the vacations of February. We discussed our scheduling and divided ourselves in two groups: Cyanolux and Bactomythril. The leaders of each group were elected: Carla (Cyanolux) and Claudia (Bactomithril).

Cyanolux: Wetlab: Simon, Seba / Drylab: Carla, Tamara, Isaac Bactomithril: Wetlab: Ulises, Bryon / Drylab: Claudia, Emilia, Max

Carla and Max were chosen to maintain the wiki updated.

Bernardo and Rolando are going to collaborate with both groups.

Also, we scheduled meetings of the whole team every Friday or Tuesday (depending on the availability of the majority of the team), and meetings with the whole team and our advisor, Professor Rodrigo Gutierrez, every other Thursday. Likewise, each group should have their own meetings.

Finally, we decided to work based on delivery dates. Each member should compromise to complete a task in specific time intervals.

March, 12 - 18, 2012

We registered our team in iGEM. Now we are officially part of iGEM 2012! Also we began to learn how to use our wiki. Our Bactomithril project is still in its initial stage, so we began to read as many papers about recombinant spider silk and protein modeling as we could.

Some of the most relevant papers we found are

- Xia et al. 2010 - “Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber”

- Widmaier et al. - 2009 - “Engineering the Salmonella type III secretion system to export spider silk monomers”

We learnt about interesting protein modeling and simulation software (e.g. Rosetta, Foldit), and some protein visualization software (PyMOL, VMD, Chimera)

Additionally, we began to send emails requesting genetic sequences from spider silk to scientists from other countries. According to what we know, in Chile nobody works with natural or recombinant spider silk proteins or its genetic sequences. So that this is a real challenge to us.

March, 19 - 25, 2012

The iGEM team Slovenia 2009 had a project potentially useful for our own project. For that reason, we sent an email to them asking if they could share their biobricks Bba_K245005 and Bba_K245113 with us. They answered us and offered their help. But their shipment never arrived. Anyway, later we realized that those parts are not fully suitable for our needs.

Also, some of us had the privilege to assist to a lecture of Andrew Hessel, an authority in Synthetic Biology from USA. Unexpectedly, he had a slide about us! (He found out about us in Internet). He said he was surprised that Chile had an iGEM team working in a forefront discipline such as Synthetic Biology.

But most importantly, we designed our first secretion constructs, consisting of a GFP Reporter Plasmid, a Protease Producer Plasmid (see section constructs for more information) and a working plasmid. This last plasmid is used as an intermediate plasmid to build the other two plasmids. We ordered the primers that we need (it takes about two weeks until they arrive!)

March 26 - April 11, 2012

We digested with restriction enzymes the HIV cleavage site, and transformed an purified the following parts of the Distribution kit 2011:

I712015 HIV 1 cleavage site I712667 HIV aspartyl protease I746908 sfGFP K103006 OmpA

We met with Ignacio Ibarra, an undergraduate student of the group of Dr. Francisco Melo. He is willing to advise us in the area of protein modeling. The problem is that spider silk proteins are semi-crystalline and is difficult to predict its structure. He recommended starting with a comparative protein analysis between our spider silk monomer of interest and other similar proteins that have its structure available in PDB. The idea is that from the structure of those similar proteins we will have a reference of the most stable conformation of our monomer. Later we should be able to improve the self-assembling of the proteins or improve some mechanical properties, such as elasticity and tenacity. Nevertheless, we were warned: protein modeling is a complex and slow task.

Recommended software: -MOE: to add hydrogen atoms. -Autodock: a suite of automated docking tools. This should be useful to evaluate the self-assembly of our monomers. -Force Field: to calculate potentials between particles. Finally, something very important happened: Christopher A. Voigt, author of “Engineering the Salmonella type III secretion system to export spider silk monomers” is going to help us with a valuable gift: a plasmid to secrete ADF-3 in salmonella. With this plasmid we can obtain the sequence of the spider silk protein ADF-3.

April 2-8, 2012

We transformed the backbone pSB1K3, so that we could amplify it when our primers arrive. Later we made and ordered the primers for our next experiment: basically the same constructs as before (GFP Reporter Plasmid) but with ADF-3 instead of sfGFP.

Ulises met with Dr. Angélica Fierro, and she accepted to be our advisor in protein modeling! Besides, we contacted Cheryl Hayashi, a well-known scientist that works with spider silk (she even spoke at TED!) [link: http://www.ted.com/talks/cheryl_hayashi_the_magnificence_of_spider_silk.html]

We asked her if she could send us the genetic sequences EF595245 and EF595246, but later we knew that those parts are not compatible with our purposes. They are fosmids and the silk sequence is only a small portion of those clones. The fosmids are low copy number and difficult to propagate. Then, it´s better to work with ADF-3.

Additionally, we wrote to Dr. Sang Yup Lee, author of one of the paper “Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber”. They produced and spun into fiber a 284.9 kDa recombinant protein of the spider Nephila clavipes in E. Coli, and we asked if he could send us his spider silk protein. Unfortunately, we didn´t receive an answer.

April 9-15, 2012

The School of Engineering at Pontificia Universidad Católica de Chile makes every year a fair of entrepreneurship and technological innovation called Ingenia. The deanship of the Faculty of Engineering is so interested in us that wants that we form part in a conversation table to spread information about iGEM and Synthetic Biology. We will have also our own stand in the fair!

On Wednesday the whole team had a meeting with Professor Rodrigo Guitérrez, and our advisors Dr. Fernán Federici (Postdoc student at Cambridge) and Dr. Mónica Vásquez (specialist in cyanobacteria). We presented them our projects and advances.

They suggested that we: - Must not forget the human practices - Should characterize existing biobricks - Should delimit and focus our projects: the time and resources are limited. - Have to consider that protein modeling is a complex task that usually takes a long time to develop.

After the meeting we decided to change our responsibilities.

Wetlab: Bryon and Max Modeling: Emilia and Ulises Wiki: Claudia

And finally our primers arrived!! (after 3 weeks). We did PCR of the different parts that we need to build our first secretion system (GFP Reporter Plasmid and the Protease Producer Plasmid). Then we had several problems with the electrophoresis, but after some attempts it worked.

5 of the 7 PCR were successfully amplified. The two parts that didn´t work are HIV cleavage site (it’s a very tiny part) and the Vector Backbone.

April 16-22, 2012

All PCR are ready!! We made our first Gibson Assembly to build the working plasmid (see constructs). We need this plasmid to build the GFP Reporter Plasmid and the Protease Producer Plasmid. But unluckily our colonies resulted to be red. This is not what we expected… later we did an analytical digestion to the DNA of those colonies, but we obtained strange results. Something went wrong.