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Team:Colombia/Journal
From 2014.igem.org
Journal
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.
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.
July 15
We got colonies from CI inverter parts 18B and 18D on 2013 distribution kit! Anddd we got a colony of IL2-3 interlab part! We are making an ON of these parts so we can miniprep tomorrow! Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.
July 16
We got colonies on tetR inverter-RFP and tetR inverter-amilCp transformation but unfortunately all our colonies are white :(…So…something is definitely not working with tetR inverter part. Things to do: IL4-3 ligation Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP
Later that day.. All the minipreps were good and arabinose 10% w/v solution was prepared to test our pBAD-LuxOD –GFP part. So in order to make the experiment on the fluorimeter tomorrow we left three ON cultures on the shaker.
July 17
Today we did digestion with SpeI and PstI to our tetR inverter part with buffer 2.1 because we need to confirm that we have the correct bps on the plasmid. We also did ligation of: tetR inverter-RFP,tetR inverter-amilCP and IL4-3 , after the ligation we transformed again, so we can have our colonies tomorrow!!! >D Amilcp, RFP and GFP digestions were confirmed on gel and tetR inverter digestion is not right, again the digested fragments do not correspond to what they are supposed to. :( We are thinking there is no hope with this part. Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).
July 18
We made the fluorescence experiment of our pBAD-LuxOD-GFP part and it didn’t work, also we did the digestion and iiiit didn’t work, so we have to make a new plan. We have to do the following: Repeat pBAD-LuxOD-GFP ligation Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN Miniprep pBAD-LuxOD-GFP and tetR inverter Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI Digest GFP with Xba-Pst
July 21
There are some parts on the registry that could work for us: The first is RBS-tetR-ter-ter-Ptet-RBS-mRFP The second is Pcat-RBS-tetR-ter-ter-Ptet-RBS-mRFP We thought maybe we could order some primers and amplify only the RBS-tetR-ter-ter-Ptet-RBS-mRFP part of the second construct orrr even better the first one is just right for us in order to add the Pqrr4 promoter amplified by PCR previously! Let’s see how it goes! Today we transformed these parts and also if this doesn’t work we will try to make it all from scratch…transforming tetR alone, ter, Ptet and RBS.
July 22
tetR protein didn’t work out. Nothing is working out :(
July 25
Digest: IL2, IL4 and IL2-3 Gel confirmation Ligate : IL2-3, IL4-3 Transform : IL2-3, IL4-3
July 28
RBS transformation and RBS-tetR-ter-ter-Ptet-RBS-mRFP in ampicilin BB didnt work out, there are some colonies in doubleTer petri dish and IL2-3 , IL4-3 have NOOO colonies at all.
We repeated IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB. Note: MilliQ Water and eppendorfs must be sterilized!!!
July 29
We have colonies of: RBS-tetR-ter-ter-Ptet-RBS-mRFP doubleTer RBS Ptet IL2-3
We repeated transformation of IL43 and left ON of the rest of the parts so we can miniprep tomorrow.
July 30
Today we are going to make digestion of all the minipreps: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI) doubleTer (EcoRI-Xba) RBS (EcoRI-PstI) Ptet (Spe-Pst) IL2-3 (Eco-PstI) IL4-3(Eco-PstI) tetR alone (Eco-Pst) And on our gel for today our lovely audience we haveeee: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI): Many many bands doubleTer (EcoRI-Xba): CONFIRMED RBS (EcoRI-PstI): CONFIRMED Ptet (Spe-Pst): CONFIRMED IL2-3 (Eco-Spe): CONFIRMED IL4-3(Eco-PstI): CONFIRMED tetR alone: CONFIRMED We made Ptet-RFP and Ptet-amilCP ligation :)
July 31
We made ON of IL43, IL23 and pBAD-LuxOD-GFP for fluorimeter Ligate RBS-tetR alone and tetR alone-doubleTer Transform Ptet-RFP and Ptet-amilCp
August 4
Miniprep Ptet-RFP, RFP, tetR-dter,amilcp,tet alone Digest RBS,RFP,amilcp,tet-dter,tet alone Ligate RBS-tetR,RBS-tet-dter Transform
August 6
Up until now we have IL1, IL23 and IL34 parts for interlab and we are waiting for our turn in the fluorimeter. Also we have tetR, tetR-dter,Ptet-RFP and Ptet-amilcp.
August 8
Digestions…digestions…sad faces…sad faces…we want to go home….we want to go home..
August 11
We are trying to do the same thing all over and over again and we are going crazy. For real.
August 12
We made ON of our transformant colonies in RBS-tetR and tetR-dt and also we did our fluorescence experiment today on IL23, pBAD-LuxOD-GFP and IL43. Results will be showed later alligatorrsss!
August 13
NOT GOOD NEWS....Our negative control (DH5alfa) gave the same results as our beloved parts on IL23, pBAD-LuxOD-GFP and IL43, soooo we will have to go over again. We did miniprep today of IL23,IL43, pSB1A3+RFP and tetR-dter. Also we digested the linearized plasmid of Amp and also tetR with Eco-Pst
Digestions: IL23 (Eco-Pst) IL43 (Eco-Pst) tetR (Eco-Spe) pSB1A3+RFP (Eco-Pst) pSB1A3 ln (Eco-Pst)
ligations: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln
Transform: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln RBS-tetR in kan plasmid pSB1C3+RFP pSB1K3+RFP
We are going to use the pSB1C3+RFP and pSB1K3+RFP plasmids for convinience, so we can see that our part inserted the plasmid by losing its color and appearing white on the petri dish.
IL23 and IL43 digestions did not work. All of the others did.
August 14
We did miniprep of pSB1C3+RFP and pSB1K3+RFP.
Our minipreps are not going good,its weird.. the concentrations are very very low and we dont know if is the lysis solution. We are going to try working with double of the amount required of lysis solution and also we are going to warm up the water in order to see if the concentration gets higher.
August 15
Today, transmitting from one computer to another!
We did miniprep of pSB1K3+RFP, pSB1K3+RFP and tetR-dTer...but once again minipreps are not working properly.
We thought today in a new possible construct, and this time the revoolutionary part is a feedback! it would be like this..
Pqrr4-rbs-tetR-Psid-Ptet-rbs-RFP-psp3
In this way we can se color much more easier and faster because the reporter is under 2 promoters that activate on the same situation.
For this we transformed--> Psid (BBa_I746364) Inducible promoter, activated by psp3 protein
RBS+Psp3 (BBa_I746351) Activator of Psid promoter
In our final month
We amplified by PCR RBS-tetR-dter-Ptet-RFP out of the registry part with pCAT. We amplified Pqrr4 for ligation with the final construct. We obtained pBAD-LUXOD-GFP and made the fluorescence measurements. We obtained the Psid-Ptet-rbs-RFP-psp3 and we are now workig on a experiment to prove that the system work transformingwith tetR protein in a different plasmid under a constitutive promoter and Psid-Ptet-rbs-RFP-psp3 in the same E.coli, growing this bacteria under different tetracicline concentrations a gradient of color should be obtained.
Also we are still trying to ligate Pqrr4 promoter to the construct Psid-Ptet-rbs-RFP-psp3 for our final results.
