We start to meet in the lab. We have been assigned a big lab in Malga, the building containing all the teaching labs. It's the biggest one. We're starting to design the constructs and primers and prepare LB medium for culture. We also make a list of all we have at our disposal and what we need to order. We're also very lucky: some thieves broke into Ross' car and stole Guzz's and Giacomo's laptops and Jason's and Guzz's apartment keys! We're not gonna sleep sweet dreams. Too bad they we're not Macs, we would have been able to retrieve them that way..

We decided to put the lab part on hiatus until monday. We realized that the powder used on day 2 was LB-agar, and not just LB, so we had to throw it away. Guzz and Ross spent the rest of the time designing primers. Giaco looked into the registry for terminators, while Andrea and Anna started taking a look protocols.

Ross and Guzz woke up early today. From Schio, they came to Trento passing through CIBio in order to retrieve DH5α e NovaBlue destinied to be grown in iGEM lab. The team then prepared LB broth and Petri dishes, and grown cells had been treated with transformation buffer and glycerol. Meanwhile, the team (flanked by a marble/concrete putto) has been involved in a brief meeting and photosession.

We transformed DH5α and Nova with pSB1K3, pSB4K5, pSB4A5 and the BioBricks for AraC pBAD (on pSB2K3) and LacI (on pSB1A2).

We found that only one Petri of five (pSB1K3) had colonies. We cultured DH5 and Nova transformed with pSB1C3 and pSB1K3. We also developed a simple Phyton tool which should allow to detect illegal sites in input sequences, returning their position if they exist and adds prefix and suffix if they does not. We're low on araC-pBAD (BBa_I0500) from the distribution kit: we're gonna need to extract it from other wells.

We looked at the feasibility of the biofilm distruption project: we optimized NucB for Caolobacter, concluding it would need synthesis, and we probably cannot afford another one. DspB could be obtained from another team (already optimized for Caolobacter). In E.coli we would need to optimize both genes and also furnish ABC transporter. The project, in the end, is discarded.

We're coming up with ideas to measure terminator efficiency and constructs designs.

//schema

We have colonies in all the plates, meaning we can now have a stock of these plasmids: we inoculate 10 colonies for each plasmid backbone. Tomorrow we'll be able to Miniprep them.

Regarding CysE, we had designed primers to PCR extract the gene from the E. coli genome. Today they arrived, so we start with the extraction.

The lacI construct is ready: we will transform BBa-Q04121, which contains lacI and a RBS and then insert a medium-strenght constitutive promoter (BBa-J23104).

//img

We're a bit confused about the araCpBAD part because of the overlaps between the regions: "the Sheref" in person will look into it.

Finally we tranform lacI from BBa-C012, araCpBAD from BBa-I0500 (both of them had failed on day 5, so we increase the DNA quantity for the transformation), plus BBa-404121 (the one we talked about previously: lacI+RBS).

The refidgerator stopped worked. Panic.

Everything is saved thanks to another fridge. Only lacI from BBa-C012 went well, which was inoculated. We then realize it would be easier to PCR extract lacI with his promoter and RBS from pET21b, a plasmid Sheref is heavily using in his lab. We design the primers with prefix and suffix so they cause an inversion of the gene: we don't want RNA pol to interfere with each other.

//schema

It's Giacomo's birthday and he didn't bring the cake. The cake is a lie.

Moar tranformations using, this time, the Registry protocol: lacI, araCpBAD (the original and those from other wells), promoters BBa_J23100, BBa-B0017 and BBa-B0010.

This time we're concentrating on the desing of the constructs fot the terminators characterization.

// spiegazione e schema (RL024A)

It's miniprepping time: pSB1K3 and pSB1C3: both gave low quantities.