Journal

May:

We started to build the main idea of our project.
15/05 → Arrival of the iGEM kit. Full of wonderful parts with an incredible varieties of functions, giving us a great push towards our goals!
17/05 - 06/06 → Development of the bioinformatic part of the project. During this period we mostly constructed our plasmid so that we could order the missing parts and start with the laboratory work.

June:

In this month we dedicated mainly to laboratory work to make practice in bacterial transformation in order to improve our results.
13/06 → First bacterial transformation of Competent cells test kit in E.coli with salin and thermal shock method; sadly first attempt failed.
15/06 → Second bacterial transformation with Competent cells test kit in E. coli with salin and thermal shock method; we failed again: it was to hot in the lab!
20/06 → Third bacterial transformation of Competent cells test kit in E. coli and this time we made all the steps in ice and IT WORKED!
22/06 → First attempt of transformation with K876011 part: unfortunately it didn’t work..!
26/06 → We didn’t give up and did the electroporation to transform our electro-competent cells: it worked!
30/06 → Finally we stored the K876011 strain in glycerol!

July:

We participated to the European meetup in Delft to expose our project and to meet all the other European teams! It has been an amazing experience and after it we started a collaboration with the CeBiTec-Bielefeld team; we decided in agreement to Skype every week so that we could learn from a more expert team as their's. We started to work on the missing plasmid, too
06-07-08/07 → European meeting in Delft, three days weren’t enough for such a beautiful experience!
11/07 → Arrival of the Gibson Assembly kit! We immediately tried it on the fragments received from IDT’s.
17/07 → first Skype with the Bielefeld Team, they gave us a ton of tips about iGEM competition since many of its aspects were hidden to us. We actually discovered how deep is the rabbit hole! In the lab we resuspended the primers we constructed and ordered to do the PCRs on IDT’s fragments and to check our first Gibson Assembly reaction’s work we did a PCR and the electrophoresis run: so we start a long series of faint stripe on the gel to fight with!
24/07 → Skype #2 with Bielefeld: they deeply explained us the function of the wiki and the complexity of building it, looks like we have a lot to think about! Also they gave us very useful advices on Human Practices, explaining us the goals and objectives of such a difficult task.
26/07 → First time we tried the elettroporation as a technique to make the bacterial transformation
31/07 → Skype #3 : discussion about the wiki, Maximilian suggested to avoid javascrit considering that it’s harder to handle and we haven’t any informatics in our team!

August:

was all about the development of the wiki and the human practices. We received two parts from Bielefeld team's project to work upon, so that we could do something in exchange for their mentoring in the frame of the ongoing collaboration. This month our project in lab was mostly frozen until the fourth week.
07/08 → Skype #4: Chris shared with us his protocol for the growth of the Escherichia coli to be engineered with their two recombinant plasmids; as soon as we receive them, we will be able to investigate the differences in the expression of the proteins encoded in their sequence. They also gave us some tips for our travel to Boston for the Jamboree!
21/08 → Skype #5: discussion about how to develop conferences for our Human Practices, calling different professors and experts to have their opinions on our project. Also, we asked many info on medal criterias and how to reach them.
21/08 – 23/08 → We did PCR on each IDT’s fragment: after each amplifications we obtained only few fragments amplificated, that we stored carefully and then we continued with the PCRs.
28/08 → Skype #6: Our fluorimeter is pretty good and can help a lot our German friends! We can take a lot of measurements during the exponential phase in an automatic way, improving a lot their data. They gave us advices on how we could resolve our problems with PCR, since it's not working for 5 of our 9 DNA fragments!!

September:

has been full of work: we reached the first good results in the lab for the Bielefeld project, and for our too. Moreover, we organized two conference calls with two professors which have been very stimulating and interesting.
04/09 – 25/09 → In this very looong period, in the lab we did a lot of PCRs on IDT’s fragment to obtain them amplificated to perform more efficiently the Gibson Assembly reaction. With hope we tried to assembly the system 1 fragments but we failed. Because we consumed the majority of the fragments, we had to re-ordered the more puzzling sequences at the IDT once again.
05/09 → Skype #7: tips for the Conference call with professor Dirk Stermerding, and about how to find others experts in our country to help us with various aspect of our project, like how to handle the public engagement and the what should be considered before writing down a survey.
18/09 → Skype #8: Team Bielefeld gave us various advices and tips to improve our PCR results, electrophoresis’s run and other practical stuff. The best part is that they shared with us the protocol for aquacloning, a pretty unexpected technique that we welcomed with arms wide open.
20/09 → Skype call with Prof. Dirk Stemerding, senior researcher at the Rathenau Instituut (NL)
25/09 → Skype #9: We must sadly announce that our third vector must be cut off from the project because we have tried all the possibilities, but seems like PCR (needed to extend the sequence lacking of some bases) won't work in any way this time. Bielefeld team shared with us a protocol to make competent cells for thermal shock, because during the summer we had some problems with our protocol CaCl2-based due to high external temperatures (the average of max temperature was over 30°C for the whole summer!)
26/09 → We experimented the acquacloning reaction: it magically worked! The day later, we carried forward acquacloning reaction with some devide (extension and ligation).
27/09 → First positive results for Bielefeld project. Conference call with David Kong about science art! David Sun Kong, Ph.D., is a Synthetic Biologist, community organizer, based in Lexington, MA. He is the Director of the MIT Media Lab's new Community Biotechnology Initiative.

October:

This month has probably been the most intense, full with the many different activities in every aspect of the competition and we couldn't be more happy with all the progresses we've made so far.
2/10 → First Meeting with an high school, located in Prato. We were a bit nervous but everything went fine. Skype #10: Brainstorming about Professor David Kong’s tips and how to apply them. The city’s blind people association could be interested to a collaboration with us, our software could give them the chance to understand lab work and sense the difference between different datas.
5/10 → Acquacloning is a success! It's a versatile and simple enzyme-free cloning approach. Acquacloning solved our assembly problems...After a month ! It's magic or maybe all genetic engineering is magical.
9/10 → Skype #11: discussion about our project, our wiki and collaborations
12/10 → Creation of vectors (e.g. pJet and pSB1C3)) and transformation of the bacteria. The transformation method is still electroporation.
14/10 → Our team went to high school Castelnuovo in Florence to present the iGem project.
18/10 → Skype #12: discussion about our medals and parts, both were complicated to deeply understand, so they checked our problems and asked their advisors too! Moreover, considering we are in the track "Art & Design", we have to pay attention to different criteria than the standard ones.
In lab, we have transformed by electroporation our plasmid and a positive control. Unfortunately, only positive control has grown but not our clone. In the evening, we extract our plasmid from the colonies transformed with pJet. It is a plasmid with large number of copies in the cell, so we ould extract the genic insert from pJet and obtained it yet amplificated without PCR!
19/10 → Plasmid extraction with kit Macherey-Nagel: no results :( But pJet is a "suicidal plasmid", and since the colonies are growing, our fragments entered!
20/10 → Castelnuovo High School students came to our labs to see the progress of our team. Everyone liked this day at the university as small scientists! These days our team also visited middle schools in Prato. The group of students was very heterogeneous but it was a great success!
23/10 → Experiment with Tecan: The multiplate reader is used to evaluate the total fluorescence present in the sample. This instrument can read fluorescence directly from multi-well plates, a highly advantageous solution for simultaneous analysis on different samples (genetic circuits). We finally got Fluorescence from the two recombinant strains we obtained!
23/10 → 24/10 Our team went to high school itis Da Vinci to present the iGem project. Many young people attended the meeting and they were very excited about it!
30/10 → Skype #13: A long lasting talk with Chris regarding how to complete the wiki with only a couple days remaining, an almost impossible task! This Skype would be the second last before Boston and the waiting is already killing us!

Team:UNIFI/Journal