Team:NGSS TR/notebook.html

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(click the months)

SEPTEMBER

September 20

iGem introduction conference conducted to students in our School, NGSS_TR team forms were distrubuted.

OCTOBER

October 10

NGSS_TR Team forms were evaluated, 2014 team is formed. Very first meeting was done, team members were illuminated about their responsibilities in the team. Followings are distributed to help team members to learn genetics and synbiology:

Idempotent Vector Design for Standart Assembly of Biobricks, Tom Knight, MIT Artificial Intelligence Laboratory

Biology, Neil A. Campbell, California University

Extreme Genetic Engineering, An Introduction to Synthetic Biology, etc, January 2007

Bio Building Basics: A Conceptual Instruction Manual for Synthetic Biology, University of California San Francisco, May 2007

BioBrik Standarts, InterTech

Sythetic Biology: New Engineering Rules for an Emerging Discipline, Molecular Systems Biology, 2006

Sythetic Biology for Artists and Designers, iGem ArtScience Team, 2009

Foundations for Engineering Biology, Drew Endy, Nature, Vol:438, November 2005

October 15

Meeting was done to discuss and learn about Sythetic Biology. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project.

October 27

Meeting was done to discuss and learn about Sythetic Biology.

NOVEMBER

November 9

First meeting with advisors from university was done.

November 19

First meeting in the university was done, project ideas were discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project.

November 28

Meetıng was done, team members chosed to specialize in the areas as following:

F. Dilara Soylu: Head of team, project research, team game;

Mariye Erol: Project research, visual modelling;

Şevval Şimşir: Video preparing

Beril Gürdap: Project research, lab experiments;

Asude Gül Akça: Project research, math modelling;

Azra Öztürk: Wiki

Verda Kılınç: Photoshop

Zeynep Bakır: Project research, math modelling;,

DECEMBER

December 6

Meeting with our advisor Fatma Betül Çevik was done, we discussed on our project ideas, some of them are provided below:

Pressure actıvated promoter, barometer

Synthetic rubber production

Restraining the conjugation

Cronometer bacteria

Timing bacteria

Air cleaning bacteria

Hemoglobin bacteria

December 15

Meeting was done to keep track of the progresses of the teams members.

December 22

Meeting was done, new project ideas discussed.

JANUARY

January 11

Team decided on wiki design, activities to do in Jamboree discussed.

January 25

Our last meeting before the 2 week school break, project ideas discussed.

FEBRUARY

11 February

First meeting after the school break, project ideas discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project.

23 February

Progresses of team members on their specialized areas discussed.

MARCH

March 8

Project ideas discussed, we decided to research on three ideas to decide on our project.

March 15

Wiki templates prepared by Azra are discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project.

March 28

We learned about disadvantages of the rapd detection systems are used to detect S Pyogenes, bacteria causes several diseases. We narrowed down our researches.

APRIL

April 4

We decided to work on a system to detect S. Pyogenes.

April 15

Construction sequence was completed and the sequence is ready for synthesize!

MAY

May 7

Meeting was done to decide on our human practice works:

Further on Project: Documentary to further explain our project.

Beyond the Lab: Funny SynBio art activities

Rock’n Twitter!: Twitter movement to introduce iGem

Colloborations: Colloborations were done with other iGem teams

May 20

Our team member Dilara had asked Genovis about sponsorhip and they accepted to provide SpeB free of charge!

May 23

SpeB enzyme arrived to the lab. We bought our flight tickets to U.S.!

May 28

Our gene sequence has arrived, we started working on them.

We transformed the gene with two compatentcells. We put RFP as a control. We put liquid culture at 11:00.

May 29

We chose three colonies from both plates and put liquid cultures. We made transformation again against the contamination possibility as the colonies in T10 were small and the ones in BL21 were large. Result: No contamination.

May 30

We isolated six liquid cultures. We could not perform digestion as the value of third colony of T10 compatentcell was low in the nano-drop. We performed the digestion and electrophoresis of the other five samples. The bands were accurate.

May 31

We examined the values that the SpeB enzyme catechol was optimized.
We tweeted #iGem2014United hashtag by having a colloboration with other iGem teams on the twitter.

JUNE

June 1

We prepared the catechol solution.

June 2

We renewed the liquid cultures.

June 3

We carried out Speb exposure test. We prepared catechol solution (dilution). We diluted our enzyme. We carried out our experiment at the 96 well plates without any catechol and by increasing the speb for control. We observed the color change through hourly-controls.

June 4

We repeated the experiment as we did not observe the yellow color yesterday. We decreased the bacteria concentration and increased the amount of pbs, the solution of the enzyme. We decided to observe the enzyme for 24 hours as it did not the stop the enzyme. We would have the results at 12:32 tomorrow.

June 5

We put catechol to the cultures waiting overnight. We perform hourly-measurements. Result: no yellow color.

June 6

June 7

June 8

We isolated the liquid cultures for control. The bands were not net, but they seemed to be accurate.

June 9

We performed isolation, digestion and electrophoresis to the liquid cultures that were put from the transformed samples of T10 and BL21. There was no ladder; and we thought the bands were accurate as they were in the same place.

We separated two blanks to check the values under spectrophotometer upon the solution of BL liquid cultures with the pbs. The total volume: 6 ml. The measurement times were minute zero, 1 hour, 2 hours, 3 hours and so on. The measurement values 0: 260 abs, 1 h: 1300 abs, 2h: -140 3 h: 180

While the epp with catechol was black, the epp without catechol was white. This indicated that the system worked, but we had to clarify it.

June 10

We repeated the experiment by changing the variables. The solution was the pbs and the total volume was 9 ml. We put enzyme. The measurement times were 0, 30 min, 1 h, 1.5 h, 2 h. We used supernatants by centrifuging the mixture before the measurement. We waited for 15 minutes for reaction with the oxygen after putting catechol. We measured values under spectrophotometer. The values were 0: -0.068, 30 min: -0.135, 1 h: 0.050, 1.5 h: -0.050 and 2 h: 0.207.

June 11

The backbone stopped. We performed ligation with the sample of BL21 liquid culture. We started the transformation. We took the plate at 8:17. We realized that it did not work as there was no reducing agent of SpeB enzyme. We chose mercaptoethanol as the reducing agent. Its molarity was not known. It would be diluted to 50-100 mM.

June 12

We directly used the mercaptoethanol as its morality was 100 Mm. We put 5% of the total volume. However, we decreased the molarity to 5 mM according to the m1.v1=m2.v2 formula. We worked with the well plate. Result: no yellow color.

We repeated the experiment with the accurate molarity. We calculated the total volume as 10 ml (1 ml catechol). We prepared 9 ml pbs mercaptoethanol mixture. It was incubated at 37 degree for 1 hour. Then, we put the catechol. Result: no yellow color. We waited overnight. We poured it to an Amp plate. We completed the transformation of the ligation sample. We took liquid cultures at 00:00.

June 13

We repeated the transformation as there was no bacteria in the liquid cultures. We took the plates at 02:00. However, we did not expect any colonies. We made a mistake with the ligation and repeated it. We diluted the mercaptoethanol to 100 mM when we realized that the molarity of the mercaptoethanol was 14.3 M. We worked as 0 enzyme and 0.4 ul enzyme for control in the well plates to calculated the time of enzyme. The measurement times were 0, 30 min, 1 h, 1.5 h, 2 h. We waited for these intervals and put catechol. The results did not change that much, and there was no yellow color.

June 14

We did the transformation of BL21+Backbone sample that we’ve done the ligation of. We solved the liquid culture with 500 ul PBS and we added 500 ul mercaptoethanol. We separated the liquid culture to four different epp and added different amounts of enzyme. We incubated at 37 degree for an hour. We centrifuged the epps and transferred the supernatants to the well plate. We added catechol and measured it at every 15 minutes. Result: we didn’t get something new.

June 15

We did the transformation again since we haven’t seen any colonies at plates. It revealed a colony at the old plates and we prepared liquid culture. We did the isolation of it.

June 16
We did digestion and electrophoresis of BB+BL isolation sample. We did the experiment again after we reduced the amount of bacteria and increased the amount of enzyme. We measured the rates at Varioskan. The experiment was repeated. At 6.20 pm, we’ll take the plate. We drew the graph of catechol, bacteria with enzyme and bacteria without enzyme at 200-600 nm.

June 17

There were no colonies at plate. We prepared four different epps.
1.      PBS+Catechol
2.      PBS+E.Coli+Catechol
3.      Catechol
4.      E.Coli+Catechol
We scanned the samples at 200-600 nm (we used spectrofotometre) and measured the rates of four different epps. We observed a changing at the epps those contains E.Coli.
We prepared two contrivances to understand what is the reason of this changing.
1.      Compatent+PBS+Mercaptoethanol+Catechol
2.      Recombinant bacterium+PBS+Mercaptoethanol+Catechol
We measured the rates at 200-600 nm. The graphs are different.
Then, we wanted to check our enzyme is working or not. We prepared 3 epps.
1.      PBS+E.Coli+Mercaptoethanol+Catechol
2.      PBS+E.Coli+0.6 ul enzyme+Catechol
3.      PBS+E.Coli+0.6 ul enzyme+ Catechol+Mercaptoethanol
We incubated the epps at 37 degree for an hour. There were no changing at the graphs so when we saw them we thought our enzyme doesn’t work. We increased the amount of enzyme to 1.2 ul and did the experiment again. There weren’t any difference.

June 18

The epps (the ones those used at yesterdays experiment) were waited overnight. We observed a color changing and measured the rates at spectrofotometre. Result: Although we didn’t see yellow color, there were changing at the graphs. The system is working.
We did lizat experiment but we didn’t get the results because the lysis buffer wasn’t working.

June 19

It revealed a colony at one of the old plates that we’ve done transformation of. We prepared liquid culture. In order to check whether our enzymes are working or not and also to control if the proteins are producing, we made the SDS-PAGE. Our system is working.