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	{{Team:UFMG Brazil/barra}} {{carlos_teste}}		{{Team:UFMG Brazil/barra}} {{carlos_teste}}
-	==Parts==	+	=Parts=
-	==Notebook==	+
-	==Protocols==	+
-	1. Solid and liquid culture media 2xYT	+
-	For 1 liter of liquid medium:	+	=Notebook=
-	- 16 g of triptone	+	==Brainstorming==
-	- 10 g of yeast extract	+
-	- 5 g NaCl	+
-	- Add ddH2O (di-deionized) to 1000 mL	+
-	For 1 liter of solid media	+	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.
-	- Same compounds as liquid medium	+	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).
-	-  3.95 grams of agar to 250 mL of liquid medium	+
-	2. Chemically competent cell preparation	+	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.
-	1. In 5 mL of 2xYT media inoculate a clone of Escherichia coli and let it grow overnight, 37°C, 180 rpm.	+
-	2. Inoculate 2 mL of E. coli culture in 200 mL of liquid culture medium in a recipient of 2 L. Grow it at 37°C, 250 rpm, until it reaches OD590 0.3 or 0.4.	+
-	3. Divide aliquots of 50 mL in 4 conical tubes and let it in ice from 5 to 10 minutes.	+
-	4. Centrifuge for 7 minutes, 4°C, 3000 rpm (~1600 x G).	+
-	5. Purge the supernatant and resuspend each pellet obtained in a recipient with 5 mL of cold solution of CaCl2.	+
-	6. Centrifuge the cells for  5 minutes, 4°C, 2500 rpm (~1333 x G). Repeat step 5 and let the cells in ice for 30 minutes.	+
-	7. Repeat step 6, but using 1 mL of cold solution of CaCl2 to resuspend the cells.	+
-	Note: In this solution, cells can stay from 12 to 24 hours.	+
-	8. Divide the cells in aliquots of 100 uL and freeze it at  -80°C.	+
-	CaCl2	+	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.
-	100 mL 500 mL	+
-	- 60mM CaCl2 0.882 g 4.4106 g	+
-	- 15% glicerol 15 mL 75 mL	+
-	- 10 mM PIPES 0.3785 g 1.8925 g	+
-	- Sterilize (autoclave) and store it in a room temperature	+
-		+
-	Note: Do not use PIPES free acid	+
-		+	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==
-	3.CoCl2 solution preparation (250 mM)	+	===January 2013===
-	- Weight  0.3246 g of CoCl2 (1 M = 129,84 g).	+	*Team UFMG was formed.
-	- Add 10 mL H2O to cobalt. Homogenize mixture.	+	*We started having meetings every Tuesday’s afternoon to discuss our team project.
-	- Filter it using a 0.22 um strainer.	+	*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.
-	4. DNA digestion	+	===February and March 2013===
-	A) Single reaction (10 uL)	+	*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.
-	- DNA 125 ng	+
-	- Buffer 10X* 1.0 uL	+
-	- BSA 10x 1.0 uL	+
-	- Enzyme* 0.25 uL	+
-	- ddH2O (di-deionized) Complete to 10 uL	+
-	* Enzymes and buffers used	+	===April 2013===
-	Enzyme Restriction site Buffer	+
-	EcoRI G↓AATC ECO	+
-	SpeI A↓CTAGT Tango 1x	+
-	XbaI T↓CTAGA Tango 1x	+
-	PstI CTGCA↓G Orange	+
		+	*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.([https://2013.igem.org/Team:UFMG_Brazil/humanpractice#Seminar])
		+	*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/.
-	B) Cobalt promoter and reporter	+	===June 2013===
		+	*The final design of our project was concluded and we received our iGEM’s biobricks kit.
-	DNA Buffer BSA 10x ddH2O Enzymes	+	===July 2013===
-	RCNA 5 uL 2 uL 2 uL 10 uL EcoRI + SpeI 0.5 uL	+
-	YFP 3.2 uL 2 uL 2 uL 11.3 uL XbaI + PstI 0.5 uL + 1 uL	+
-	Plasmid 10 uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL	+
-	C) TorCAD and Chloramphenicol plasmid resistance (PSB1C3)	+	*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. ([https://2013.igem.org/Team:UFMG_Brazil/humanpractice#Videos]).
		+	*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 ([https://2013.igem.org/Team:UFMG_Brazil/humanpractice#The_Brickard_Game]).
-	DNA Buffer Orange 10x BSA 10x ddH2O Enzymes	+	*We succeded in preparing our first construct after cloning of RCNA+YFP into PSB1A3.
-	- PSB1C3 25 ng/uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL	+
-	- TorCAD 10 ng/uL 2 uL 2 uL 5 uL EcoRI + PstI 0.5 uL	+
-	- 37°C for 4 hours	+	===August 2013===
-	5. Ligation	+	*We  started fluorimetric assays with bacteria carrying the plasmid construct  RCNA+YFP, to verify the effect of  cobalt in the expression of YFP.
-	A) RCNA-YFP-PSB1A3	+	*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.
-	- Plasmid 2 uL	+	===September 2013===
-	- RCNA 2.5 uL	+
-	- YFP 2.5 uL	+
-	- Buffer 10x 1 uL	+
-	- T4 DNA ligase 1 uL	+
-	- ddH2O (di-deionized) 1 uL	+
-	- Incubate it overnight at 4ºC	+
-		+	*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” ([https://2013.igem.org/Team:UFMG_Brazil/humanpractice#Videos]).
		+	*On September 21th an interview with our team was published in one of the largest newspaper in the country, “Estado de Minas” – [https://2013.igem.org/Team:UFMG_Brazil/humanpractice#In_the_news].
-	B) PSB1C3 and TorCAD (10uL)	+	*27th September: WIKI FREEZE!!!!
-	- Plasmid 2 uL	+
-	- TorCAD 2.5 uL	+
-	- Tampon 10x 1.0 uL	+
-	- T4 DNA ligase 1 uL	+
-	- ddH2O (di-deionized) 3.5 uL	+
-	- Incubate it overnight at 4ºC	+
		+	===October 2013===
-	6. Transformation Protocol	+	*Regional Jamboree in Chile.
-	1. Start thawing the chemically competent cells on ice.	+	*Meeting in Hackathom event in Minas Gerais state:"Synthetic Biology and Programming of Innovative Biological Systems".
-	2. Add 1 - 2 uL of DNA to the 2 mL tube. Pipet up and down a few time, gently. Make sure to keep the competent cells on ice.	+	*Final Experiments.
-	3. Close the tubes and incubate the cells on ice for 30 minutes.	+	*28th October: WIKI FREEZE!!!!
-	4. Add cells tubes by immersion in a preheated water bath at 42 °C for 60 seconds.	+
-	5. Incubate the cells on ice for 5 minutes.	+
-	6. Add 400 uL of 2xYT media (make sure that the broth does not contains antibiotics and is not contaminated) to each transformation.	+
-	7. Incubate the cells at 37°C for 1 hour while the tubes are rotating or shaking.	+
-	Important: 2 hours recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.	+
-	8. Label two petri dishes with 2xYT agar (AMP or CHL). Plate 20 uL and 200 uL of the transformation onto the dishes, and spread. This helps you ensure that you will be able pick out a single colony.	+
-	9. Incubate the plates at 37°C for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start break down and untransformed cells will begin to grow, because the resistance enzyme will be excreted by the bacteria inactivating the antibiotic outside of it.	+
-	10. Pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.	+
-		+	===November 2013===
		+	*World Championship Jamboree.
-	7. Miniprep
-
-	- We used Promega and Invitrogen kits. We followed manufacturer’s indications .
-
-
-
-
-
-
-
-
-
-
-	8. PCR Protocol
-
-	Compound Volume Program Cilcles Repeat
-	1. ddH2O 8 uL - - -
-	2. Buffer IB 10x 1.5 uL - 10 min -
-	3. dNTP’s 2.5 mM 1.5 uL 94 °C 1 min 1x
-	4.. Primer VF2 10 uM 0.4 uL 94 °C 1 min 30x
-	5. Primer VR 10 uM 0.4 uL 50 °C 1.5 min 30x
-	6. Taq 5u / uL 0.2 72 °C 10 min 30x
-	7. DNA 3 uL 72 °C 10 min 1x
-	8. Final volume 15 uL 4° C - -
-
-	Note:  Using a thermocycling on steps 3 to 7
-
-	9.  Fluorimetric assay for RCNA-YFP activation
-
-	1. Measure OD 600 of cultures to be assessed.
-	2. Dilute cultures to OD 600 = 0.02, in a volume of 3 mL.
-	3. Prepare a solution of CoCl2  1 mM, using 6 uL of 250 mM solution ( look protocol 3) and 1494 uL of 2xYT media.
-	4. Make other 6 solutions using the mixture on step (3).
-	50 uM -> 50 uL solution + 950 uL of media 2xYT
-	100 uM -> 100 uL solution + 900 uL of media 2xYT
-	150 uM -> 150 uL solution + 850 uL of media 2xYT
-	200 uM -> 200 uL solution + 800 uL of media 2xYT
-	250 uM -> 250 uL solution + 750 uL of media 2xYT
-	300 uM -> 300 uL solution + 700 uL of media 2xYT
-	5. Add 100 uL of the cultures from step (2) in each well completing 21 wells. Separating appropriately the cultures in triplicate, add to each column a different solution from step (4).
-	6. Add 100 uL of media 2xYT in another wells completing 21 wells, to discard the noise from media
-	7. Prepare the designed wells from step (5) adding 100 uL of cobalt solution from step (3)
-	8. Seal the plaque
-	9. Read fluorescence: 514 nm (excitation) and 527 nm (emission), every 15 minutes, for 16 hours.
-
-	Note: It is not recommended read absorbance using black plaque as it causes interference on reading, despite of it is better for fluorimetric assay. Instead of it, use transparent plaques.
-
-	==Safety==
-	===Course===
-	<html><center><iframe width="960" height="650" src="//www.youtube.com/embed/_vb1huQtiwk" frameborder="4" allowfullscreen></iframe></center> </html>
-
-	===Hazard===
-	===Provision===
-
-	==Experiments==
-	==Results==
	{{Team:UFMG Brazil/sponsor}}		{{Team:UFMG Brazil/sponsor}}

---

Latest revision as of 15:09, 27 October 2013

Contents 1 Parts 2 Notebook 2.1 Brainstorming 2.2 Day by Day 2.2.1 January 2013 2.2.2 February and March 2013 2.2.3 April 2013 2.2.4 May 2013 2.2.5 June 2013 2.2.6 July 2013 2.2.7 August 2013 2.2.8 September 2013 2.2.9 October 2013 2.2.10 November 2013

Parts

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 Hackathom event in Minas Gerais state:"Synthetic Biology and Programming of Innovative Biological Systems".
• Final Experiments.
• 28th October: WIKI FREEZE!!!!

November 2013

• World Championship Jamboree.

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