June 24

Today was the first day at the lab!! Finally! After finding out the parts we ordered from the registry had been sitting in a locker for the last two weeks and managing to salvage them we re finally up and running. First thing first, today we are making electrocompetent cells and transforming our first parts. We are using TOP 10 strain to make our transformations. We are transforming mRFP (BBa_K518012), pBAD (BBa_K206000), tetR inverter (BBa_Q04400), amilCp (BBa_K592025), pBAD-CqsA (BBa_K581011), pTet-LuxOD47E (BBa_K218017), pqrr4-GFP (BBa_K581009) and LuxU-CqsS-LuxO (BBa_K581010). We are starting with the Interlab project, so we are transforming those parts as well. For convenience, we have decided to name the Interlab parts IL1 (BBa_I20260, promoter+GFP in 1K3), IL2 (BBa_J23101, promoter), IL3 (E0240, GFP) and IL4 (BBa_J23115, promoter).

We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.

June 25

We have transformants! Well, almost. Nothing grew on the IL4 plate :(. But nothing to worry about, we are just repeating the transformations. We made passes to liquid media of single colonies of all the other parts and we are hoping to do miniprep of all them tomorrow.

We also cryopreserved all the bacteria we got from the registry. Just made a fresh new Kanamycin stock, so we can now transform tetR inverter.

June 26

Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.

We just realized the tetR inverter part has two other twins in the registry. We are transforming those as well and hope one of them works.

June 27

Today we prepared homemade lysis buffer for our minipreps, but apparently it wasn't very effective. We could not see any DNA after running our miniprerps in a gel.

July 1

After a long weekend, we are back in business. We have colonies in all the plates with the trnasformations of the tetR inverter! We made passes of all three parts to liquid LB for miniprep tomorrow.

July 2

We repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!

July 3

Now that we have all the parts, we can start building our construct! We are starting with the Interlab parts for now. Today we digested both promoters and GFP , ligated them and transformed them using our electrocompetent TOP 10 E. coli.

July 4

All our transformations were unsuccessful. After running the digestions in a gel we realized that the digestion was not complete. We did some positive controls on all the enzymes to verify that they are working properly, but it all seems to be working fine.

July 7

Today we are measuring fluorescence of the first part of the Interlab Study! We are also trying to begin joining the first two parts of our construct tetR inverter and mRFP. Here is some doodles we used to explain to the new iGemers how the process of digestion and ligation works and the logic behind it.

PONER FOTO 1

July 8

Primers have arrived! We are resuspending our primers and using them to make our first PCRs of the year. We are trying to get pqrr4 and LuxOD47E.

PCR products have just been confirmed with gel electrophoresis! Finally something is working!

July 9

Ligation and transformation of the two remaining Interlab parts was a complete failure. We decided to purify the GFP fragment from the digestion and try using this to make the ligation. Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.

July 10

Today we digested all the parts with the appropiate enzymes to make the constructs from yesterday. We ligated and trasnformed them. We also decided to start working on tetR inverte-mRFP and tetR inverter-amilCp.

July 11

No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.

Jul 14

We have colonies this time, but they are white. We were expecting red and blue colonies. After verifying the tetR inverter parts we got from the distribution kit, we found out that the size of the digested fragments do not correspond to the 902 bp or the 2070 bp of the inverter and pSB1C3. All three parts look the same. It seems we are not going to be able to use them.

We are now looking into the possibility of using CI inverter instead of the tetR invertes. We are transforming the two CI invertes from the distrbution kit. Hopefully we'll better luck this time.

We have not been able to get the two remaining Interlab constructs.