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Notebook

Brainstorming

In the beginning, we decided to meet weekly to discuss ideas to be developed in iGEM competition. We scheduled a fixed day and time for such discussion and the presence of all members was considered mandatory, as we had in mind that a good project would require the involvement of everybody.

So, based on this perspective and considering that most of our colleagues have a knowhow applied to human health, we thought about tropical diseases, like dengue. We had imagined what we could do to precociously diagnose that disease as a measure to provide a fast and precise health care to patients positively diagnosed. Summarizing, we have firstly considered to develop a fast diagnostic tool for dengue, which everyone could use without restrictions, based on a GMO (Genetically Modified Organism).

Despite having made a big effort to implement it, many factors turned it an unviable project. First, we didn’t have means to deal with the vector, Aedes aegypti, and we couldn’t establish a viable way to use a GMO to our primary purposes. To try to solve these problems, we invited a researcher from Funed (Fundação Ezequiel Dias - Brazil), Alzira Batista Cecilio, to talk to us about the disease and the fast diagnostic test that she was developing in her studies. This gave us some possibilities, but all of them were too complex to be applied to iGEM in short time. We kept working on building new ideas to be implemented.

In order to perform a search, we divided our team in groups, which were encouraged to give new and viable ideas to be developed. One of them had a performable idea: check for biomarkers in order to precociously diagnose heart diseases, a priori based in choline detection. But, as this substance is released to blood flow in response to many disturbs, we thought that more biomarkers would be necessary in order to provide a reliable diagnosis. Occurred to us that it would be interesting to add a biomarker already validated and well described. We thought of using creatine-kinase MB (CK-MB), but it does not have an useable receptor or inducible promoter available, at least one that we could find, to be expressed on our chassis. Troponin was cogitated to be another of our relevant biomarkers, however, it has also shown to be unviable due to the absence of a receptor we could use and or a channel to transport it into the cell.

After an intense search, in the end we have agreed to use three biomarkers: Brain Natriuretic Peptide (BNP), Trimethylamine-N-Oxide (TMAO) and Ischemia Modified Albumin (IMA). BNP is a validated biomarker for Acute Coronary Syndrome (ACS) and it has a receptor that could be used to detect BNP, despite of huge size of its receptor (NPR-A) which have a transmembrane site. TMAO, which is not considered a validated biomarker, but it came out in our latest searches it could be used as a heart failure predictor, since this substance attacks the heart muscle tissue and provokes necrosis, the main factor of myocardial infarction. IMA is an indicative of any sort of ischemia and it was validated by FDA as a biomarker for ACS, although it is best used as a negative predictor than a positive one (meaning that its absence indicates that everything is probably fine, but its presence just hints that there might be something wrong).

Day by Day

January 2013

Team UFMG was formed.
We started having meetings every Tuesday’s afternoon to discuss our team project.
Introductory presentations were given by members of the team to explain to the group the basic concepts involved in the iGEM competition, what Biobricks are, how computer science and biology can work together to create new living things, etc.

February and March 2013

We discussed previous projects developed by iGEM teams and each team member was asked to bring new ideas for our project. After several presentations and discussions, we select the main theme of our project: cardiovascular diseases biomarkers.

April 2013

On April 19th we presented our project to various Professors and graduate students from the Biochemistry and Immunology Department at UFMG. During these discussions we had the opportunity to present our initial ideas and discuss them with people that were not directly involved with the project. These discussions were very important since we received important feedback that helped us to improve designing our final proposal.([1])
During all meetings the group had during this month, we discuss the literature about cardiac diseases biomarkers, in order to improve our project.

May 2013

Our project has been improved and the definition of the biomarkers that we were going to assay became more and more clear.
We started putting in place our ideas about the human practice components of our project.
We published a text about our project in the SynbioBrasil’s blog, a blog created by the iGEM team from USP, see on: http://synbiobrasil.org/2013/05/28/minas-gerais-no-igem/.

June 2013

The final design of our project was concluded and we received our iGEM’s biobricks kit.

July 2013

We started our experiments by trying to grow the bacteria containing the plasmids, which was quite difficult because we had trouble with the cloramphenicol that we were using (it was expired and didn’t work well ). After solving that problem we were able to grow the cells and purify our plasmids.
Biosafety Practices: we concluded an one-week course with Neuza Antunes about laboratory safety practices. ([2]).
As part of the human Practices component of our project, we had a wonderful experience participating in the course UFMG & Escolas. This is a program that is being developed for many years in our University and has the goal of bringing high school students as well as school teachers to our campus to let them develop research projects according to their interests and curiosities. Teaching synthetic biology to those children and teenagers was quite enlightening. We used our Brickard Game to make it more attractive ([3]).

We succeded in preparing our first construct after cloning of RCNA+YFP into PSB1A3.

August 2013

We started fluorimetric assays with bacteria carrying the plasmid construct RCNA+YFP, to verify the effect of cobalt in the expression of YFP.
We used the oligonucleotide that we had asked to be synthesized containing sequences of the TorCAD promoter and tried to clone this sequence into PSB1C3.
We perform PCR and restriction enzyme digestion to confirm the identity of the constructs PSB1A3_RCNA+YFP and PSB1C3_TorCAD.

September 2013

Additional fluorimetric assays were perfomed with bacteria transformed with PSB1A3_RCNA+YFP using different cobalt concentrations and in the presence of sera from normal mice or ischemic mice.
We tried to clone the TorCAD promoter upstream RFP into the PSB1C3 plasmid.
Our new biobricks were sent to iGEM Headquarters.

We created “The E. coli Dilemma video” ([4]).
On September 21th an interview with our team was published in one of the largest newspaper in the country, “Estado de Minas” – [5].

27th September: WIKI FREEZE!!!!

October 2013

Regional Jamboree in Chile.
Meeting in Hackathon event in Minas Gerais state:"Synthetic Biology and Programming of Innovative Biological Systems".
Final Experiments.
28th October: WIKI FREEZE!!!!

November 2013

World Championship Jamboree.

Parts Issue

We tried to order the synthesis of these sequences:

 PromTorCAD (125 bp) :  
 5'-CGAACGAATTCGCGGCCGCTTCTAGAGATTCTGTTCATATCTGTTCATATTCCGTTCATCCTGACCA
 GTGCCGCTGTTCATATTTGCTCATTAAGATCGCTTCATACTAGTAGCGGCCGCTGCAG - 3'

 PromPDE5_OM (180 bp):
 5'-GAATTCGCGGCCGCTTCTAGGGCCGCGCGGCCTCCTTCCAGCCGCCGCCACTTGGCTTCCGGAG
 AGCTCGCCGGGCGCTGCCGCCGCCGCCGCCGCCGCCTCCTGGGAACCAGGGGACTGAAGAGCCTG
 CGAGAGCGGAACACTGCCGGACCCCGGGTGTACTAGTAGCGGCCGCTGCAG - 3'

 PromPDE5_OX (275 bp): 
 5'-GAATTCGCGGCCGCTTCTAGGCCGCCGCCGCCGCCGCCGCCTCCTGGGAACCAGGGGACTGAAG
 AGCCTGCGAGAGCGGAACACTGCCGGACCCCGGGTGGGGGGGCGCAGCAGCTGCGCCTGGCCCCG
 CCCACCACACCTGGGCGCCCGTAGAACCGCGCGGGGCGGGGCGGGGCAGGAGGCTGGCCTGGCGC
 TCCGGCCGCTTTGTCGAAAGCCGGCCCGACTGGAGCAGGACGAAGGGGGAGGGTCTCGAGTACTAG
 TAGCGGCCGCTGCAG - 3'

 NPRA_1 (473 bp): 
 5’-GAATTCGCGGCCGCTTCTAGATGCCGGGTCCGCGTCGTCCGGCGGGTTCTCGTCTGCGTCTGCTG
 CTGCTGCTGCTGCTGCCGCCGCTGCTGCTGCTGCTGCGTGGTTCTCACGCGGGTAACCTGACCGTTG
 CGGTTGTTCTGCCGCTGGCGAACACCTCTTACCCGTGGTCTTGGGCGCGTGTTGGTCCGGCGGTTGA
 ACTGGCGCTGGCGCAGGTTAAAGCGCGTCCGGACCTGCTGCCGGGTTGGACCGTTCGTACCGTTCT
 GGGTTCTTCTGAAAACGCGCTGGGTGTTTGCTCTGACACCGCGGCGCCGCTGGCGGCGGTTGACCT
 GAAATGGGAACACAACCCGGCGGTTTTCCTGGGTCCGGGTTGCGTTTACGCGGCGGCGCCGGTTGG
 TCGTTTCACCGCGCACTGGCGTGTTCCGCTGCTGACCGCGGGTGCGCCGGCGCTGGGTTTCGGTGT
 TAAAGACGAA-3’

 NPRA_2 (497 bp):
 5’-CCGGCGCTGGGTTTCGGTGTTAAAGACGAATACGCGCTGACCACCCGTGCGGGTCCGTCTTACGC
 GAAACTGGGTGACTTCGTTGCGGCGCTGCACCGTCGTCTGGGTTGGGAACGTCAGGCGCTGATGCTG
 TACGCGTACCGTCCGGGTGACGAAGAACACTGCTTCTTCCTGGTTGAAGGTCTGTTCATGCGTGTTC
 GTGACCGTCTGAACATCACCGTTGACCACCTGGAATTTGCGGAAGACGACCTGTCTCACTACACCCG
 TCTGCTGCGTACCATGCCGCGTAAAGGTCGTGTTATCTACATCTGCTCTTCTCCGGACGCGTTCCGTA
 CCCTGATGCTGCTGGCGCTGGAAGCGGGTCTGTGCGGTGAAGACTACGTTTTCTTCCACCTGGACAT
 CTTCGGTCAGTCTCTGCAAGGTGGTCAGGGTCCGGCGCCGCGTCGTCCGTGGGAACGTGGTGACGG
 TCAGGACGTTTCTGCGCGTCAGGCGTTCCA – 3’

 NPRA_3 (497 bp):
 5’-TCAGGACGTTTCTGCGCGTCAGGCGTTCCAGGCGGCGAAAATCATCACCTACAAAGACCCGGACA
 ACCCGGAATACCTGGAATTTCTGAAACAGCTGAAACACCTGGCGTACGAACAGTTCAACTTCACCAT
 GGAAGACGGTCTGGTTAACACCATCCCGGCGTCTTTCCACGACGGTCTGCTGCTGTACATCCAGGCG
 GTTACCGAAACCCTGGCGCACGGTGGTACCGTTACCGACGGTGAAAACATCACCCAGCGTATGTGGA
 ACCGTTCTTTCCAGGGTGTTACCGGTTACCTGAAAATCGACTCTTCTGGTGACCGTGAAACCGACTTC
 TCTCTGTGGGACATGGACCCGGAAAACGGTGCGTTCCGTGTTGTTCTGAACTACAACGGTACCTCTC
 AGGAACTGGTTGCGGTTTCTGGTCGTAAACTGAACTGGCCGCTGGGTTACCCGCCGCCGGACATCCC
 GAAATGCGGTTTCGACAACGAAGACCCGG – 3’

 NPRA_4 (495 bp):
 5’-CGAAATGCGGTTTCGACAACGAAGACCCGGCGTGCAACCAGGACCACCTGTCTACCCTGGAAGTT
 CTGGCGCTGGTTGGTTCTCTGTCTCTGCTGGGTATCCTGATCGTTTCTTTCTTCATCTACCGTAAAATG
 CAGCTGGAAAAAGAACTGGCGTCTGAACTGTGGCGTGTTCGTTGGGAAGACGTTGAACCGTCTTCTC
 TGGAACGTCACCTGCGTTCTGCGGGTTCTCGTCTGACCCTGTCTGGTCGTGGTTCTAACTACGGTTCT
 CTGCTGACCACCGAAGGTCAGTTCCAGGTTTTCGCGAAAACCGCGTACTACAAAGGTAACCTGGTTG
 CGGTTAAACGTGTTAACCGTAAACGTATCGAACTGACCCGTAAAGTTCTGTTCGAACTGAAACACATG
 CGTGACGTTCAGAACGAACACCTGACCCGTTTCGTTGGTGCGTGCACCGACCCGCCGAACATCTGCA
 TCCTGACCGAATACTGCCCGCGTG – 3’

 NPRA_5 (491 bp):
 5’-TCTGCATCCTGACCGAATACTGCCCGCGTGGTTCTCTGCAAGACATCCTGGAAAACGAATCTATCA
 CCCTGGACTGGATGTTCCGTTACTCTCTGACCAACGACATCGTTAAAGGTATGCTGTTCCTGCACAAC
 GGTGCGATCTGCTCTCACGGTAACCTGAAATCTTCTAACTGCGTTGTTGACGGTCGTTTCGTTCTGAA
 AATCACCGACTACGGTCTGGAATCTTTCCGTGACCTGGACCCGGAACAGGGTCACACCGTTTACGCG
 AAAAAACTGTGGACCGCGCCGGAACTGCTGCGTATGGCGTCTCCGCCGGTTCGTGGTTCTCAGGCG
 GGTGACGTTTACTCTTTCGGTATCATCCTGCAAGAAATCGCGCTGCGTTCTGGTGTTTTCCACGTTGA
 AGGTCTGGACCTGTCTCCGAAAGAAATCATCGAACGTGTTACCCGTGGTGAACAGCCGCCGTTCCGT
 CCGTCTCTGGCGCTGCAATCT – 3’

 NPRA_6 (495 bp):
 5’-CCGTTCCGTCCGTCTCTGGCGCTGCAATCTCACCTGGAAGAACTGGGTCTGCTGATGCAGCGTTG
 CTGGGCGGAAGACCCGCAGGAACGTCCGCCGTTCCAGCAGATCCGTCTGACCCTGCGTAAATTCAA
 CCGTGAAAACTCTTCTAACATCCTGGACAACCTGCTGTCTCGTATGGAACAGTACGCGAACAACCTG
 GAAGAACTGGTTGAAGAACGTACCCAGGCGTACCTGGAAGAAAAACGTAAAGCGGAAGCGCTGCTGT
 ACCAGATCCTGCCGCACTCTGTTGCGGAACAGCTGAAACGTGGTGAAACCGTTCAGGCGGAAGCGTT
 CGACTCTGTTACCATCTACTTCTCTGACATCGTTGGTTTCACCGCGCTGTCTGCGGAATCTACCCCGA
 TGCAGGTTGTTACCCTGCTGAACGACCTGTACACCTGCTTCGACGCGGTTATCGACAACTTCGACGT
 TTACAAAGTTGAAACCATCGGTGACGCG – 3’

 NPRA_7 (459 bp):
 5’-GTTTACAAAGTTGAAACCATCGGTGACGCGTACATGGTTGTTTCTGGTCTGCCGGTTCGTAACGGT
 CGTCTGCACGCGTGCGAAGTTGCGCGTATGGCGCTGGCGCTGCTGGACGCGGTTCGTTCTTTCCGTA
 TCCGTCACCGTCCGCAGGAACAGCTGCGTCTGCGTATCGGTATCCACACCGGTCCGGTTTGCGCGG
 GTGTTGTTGGTCTGAAAATGCCGCGTTACTGCCTGTTCGGTGACACCGTTAACACCGCGTCTCGTATG
 GAATCTAACGGTGAAGCGCTGAAAATCCACCTGTCTTCTGAAACCAAAGCGGTTCTGGAAGAATTTGG
 TGGTTTCGAACTGGAACTGCGTGGTGACGTTGAAATGAAAGGTAAAGGTAAAGTTCGTACCTACTGGC
 TGCTGGGTGAACGTGGTTCTTCTACCCGTGGTTAATACTAGTAGCGGCCGCTGCAG – 3’

What are this sequences?

PromTorCAD:

- Promoter indirectly sensitive to TMAO.

PromPDE5_OM:

- Promoter of PDE5 (phosphodiesterase 5), sensitive to cGMP. It was going to be used to indirectly detect BNP.

PromPDE5_OX: