Team:UC Chile/Cyano/Notepad/week20

From 2012.igem.org

Monday: Today is suppose to be a holiday, I'm not really sure why, but the team is still working. The building seems abandoned and we are the only ones in here muajuajuajuajua (evil laughter).

About our work in progress: we have our second arduino project. It is a photoresistor able to measure the intensity of light inputs. The arduino program is linked to old Juano's c# aplication so we can see a graph of the data in real time. We have only tried it with LEDs so far and the system proved to be sensible enough. The next step is to measure bacterial biolumniscence. We hope to do so tomorrow. We transformed e. coli with lux brick (at race speed) in order to have a really nice shining flask and check if our arduino system can sense bacterial produced light properly.

Meanwhile in the wetlab... We built the following parts trough a ligation protocol. Then we transformed and plated e.coli.

1. RS1 + KanR + psb1C3 . Plate resistance: chloramphenicol and kanamycin.
2. RS1 + KanR. Plate resistance: chloramphenicol.
3. RS1+ KanR. Plate resistance: chloramphenicol and kanamycin.
4. B0014 + RS2 + psb1C3. Plate resistance: chloramphenicol.
5. B0014 + RS2. Plate resistance: kanamycin.

Also, we did an electrophoresis run of LuxCDEG PCR product. Nothing showed up u.u. But we could purify more psB1C3 (it does not make up for lux though)

Finally, we prepared some parts to send to our international advisor (yeah, the Argentinian) so he can try to assemble them by the Gibson technique.

SIMON LEFT US TODAY :_( He'll be spending the next three (yes, THREE!) weeks in San Francisco. He couldn't help but trade us for sunny californian beaches...

Tuesday: We had a videoconference with Copenhagen and Valencia teams. As we are working in very similiar projects we decided to establish a collaborative alliance. Eventually, we will share our constructs to compare their performance in the different systems. Go teams go!!!

E. colis with lux brick were grown in LB media. L-Arabinose 2 mM was added. The sensitivity test for the arduino circuit will be done tomorrow.

Later on we checked yesterday's plates and all of them had colonies (yay!!), although the ones with newer ligation buffer had better yield (plates 1 and 5). The previous low transformation efficiencies might have been because of this. We culture colonies from plates 1 and 5 in bacterial tubes for further extraction.

Another gel run for LuxCDEG PCR product was done. Again, no sign of bands. Run for LuxCDEG with X+P also showed nothing. We think we must have done something incorrectly. We'll try the following ligation: (Lux CDEG1 E+S digestion) + (Lux CDEG2 X+P digestion) + (psB1C3 E+P).

As a day with no transformations is a dull one we transformed: Lux CDEG in psB1C3, Lux CDEG in psB1A2 and Lux CDEG in psB1A2 (from plasmid).

Checking final details of synechocystis transformation protocol. We hope to do it on Thursday!

Parts for our Argentinian advisor are on their way to Cambridge :) hope they make it through customs!

Wednesday:

The bacteria with the last ligation didn't grow in kanamycin plates. We believe the digested parts were wrong labeled (or we just didn't understand Simon's label system). Therefore, a PCR colony was run and gave negative results. Decided to digest parts Lux CD and Lux EG + terminator again.

One liter of liquid BG11C media and two schott bottles with 1/2 liter each of BG11C solid were prepared. Expecting to grow our synechocystis soon :)

Colony PCR of bacteria with RS2+B0014+psB1C3 turned out to be negative so a massive colony PCR was run (21 colonies!) corresponding to the kanamycin plate. All PCRs were negative. Could be the primers failing?

PCR of parts Lux CD and Lux EG could not be distinguised, probably because they are the same size as the backbone. Anyhow the parts will be ligated and then transformed.

Thursday:

Lux CD and Lux EG were ligated and then transformed. Everything is being settled for tomorrow's synechocystis transformation.

We took some beautiful photos of our Lux bacteria flask :D!!! Al seems really creepy in there, although Simon believes Al is just trying to hug his new friends (message sent through facebook).

Finally we could try the arduino system and nope, it's not sensitive enough. We'll wait for the sensor which arrives on Monday. And Carla left today (everyone is leaving us...). She'll be spending the next week in her homeland beerland.



Friday: Synechocystis transformed with: psb1A3_Int C with RFP (from Utah State team) and psbAB+GFP. DNA concentration and volume: 1 ug/uL in 10 uL.

Multi colony PCR of C4 and Lux CDEG consisting of 10 colonies was run. Another four PCRs for B0014+RS2+KanR and one for psB4K5+sfGFP were also run. Lux resulted in two bands and colony PCRs gave several sizes.

Saturday: Terminator B0014 will be replaced for B0015 as we've been having difficulties with the first part. Synechocystis were changed to plates with half amount of antiobiotic according to protocol. New colony PCR for LuxCDEG and B0014+RS2 positive colonies. New RS2 E + P was obtained and then religated with B0015. E. coli were transformed and plated with the part.

Sunday: Yesterday's PCR was run and strange bands appeared (again). New strategy: will purify RS2 and B0014 and B0015, cut an will try again as such.

Also [RS1+Kan](E+S) + [RS2] (X+P).... Lux CD+ Lux EG to int_C, and PCR Lux CDEG Plasmid.

Purifications:

-RS1+Kan (E+S)
-RS2 (X+P)
-B0014 (E+S)
-B0015(E+S)

Then, the following ligations were made:

-(Rs1+Kan)+RS2 in psb1A2
-RS2+ B0014 in psb1C3
-RS2+ B0015 in psb1C3
-LuxCD+LuxEG in Int_C

PCR Lux CD and Lux EG with VF and VR or SuffixR_digest and PreffixF_digest. Result, Lux CD vary faint and LuxEG looks cool. Bands were extracted.