Thursday June 30, 2016
Saturday July 2, 2016
Monday July 4, 2016
Tuesday July 5, 2016
Wednesday July 6, 2016
Thursday July 7, 2016
Friday July 15, 2016
Saturday July 16, 2016
Monday July 18, 2016
Tuesday July 19, 2016
Wednesday July 20, 2016
Thursday July 21, 2016
Friday July 22, 2016
Saturday July 23, 2016
Tuesday July 26, 2016
Wednesday July 27, 2016
Thursday July 28, 2016
Friday July 29, 2016
Monday August 1, 2016
Tuesday August 2, 2016
Wednesday August 3, 2016
Thursday August 4, 2016
Friday August 5, 2016
Tuesday August 9, 2016
Thursday August 11, 2016
Friday August 12, 2016
Saturday August 13, 2016
Monday August 15, 2016
Tuesday August 16, 2016
Wednesday August 17, 2016
Thursday August 18, 2016
Friday August 19, 2016
Saturday August 20, 2016
Sunday August 21, 2016
Monday August 22, 2016
Tuesday August 23, 2016
Wednesday August 24, 2016
Thursday August 25, 2016
Saturday August 27, 2016
Monday August 29, 2016
Tuesday August 30, 2016
Wednesday August 31, 2016
Thursday September 1, 2016
Friday September 2, 2016
Saturday September 3, 2016
Sunday September 4, 2016
Monday September 5, 2016
Friday September 9, 2016
Saturday September 10, 2016
Monday September 12, 2016
Tuesday September 13, 2016
Wednesday September 14, 2016
Friday September 16, 2016
Saturday September 17, 2016
Sunday September 18, 2016
Monday September 19, 2016
Tuesday September 20, 2016
Wednesday September 21, 2016
Thursday September 22, 2016
We tested our antibiotics: ampicillin, kanamycin, chloramphenicol with our PLYS and Shuffle bacterias
We repeated the testing of our antibiotics and we add BL21 to the experiment.
Transformation (Shuffle calcium competent E.coli). The parts used were: GFP [AMP] (11P from the 2014 IGEM kit plate #4), Promotor (17P from the 2015 IGEM kit plate #4). There was no growth at all (even in our control). Probably the heat shock wasn’t done correctly (1.20 seconds, 42°c instead 0.50 s) . We reactivated our strains Top10 and DH5-alpha.
We transferred our strains of bacteria to a new culture media LB. We made some experiments to see if our CAM was working.
We made transformation of the following strain: Top10 with GFP and also another one with DH5-Alpha with GFP.
The only strain that was transformed was Top10 with times 40 sec and 60 sec. We also made the solutions we needed for MiniPrep Solution I, II and III that will be used in the rest of the project. A digestion was also made with ECORI and Xbal.
An electroforesis gel was also made.
Digestion of the Minipreps of P1, P2, G2 and G4, using PstI and XbaI. Electrophoresis of both digestions and minipreps.
Electrophoresis of the minipreps. We used two different ladders: two-log and Axigen 100 bp. Purification of the gel with Wizard SV Gel and PCR Clean-up System from Promega. We measured the concentration of our DNA in NanoDrop. Ligation. We made electrocompetent cells.
We tested our ligations transforming BL21 cells by heat shock. AMP, there was no growth in the petri dishes inoculated with them.
Acidithiobacillus ferrooxidans was obtained from the Geomicrobiology Laboratory of the Autonomous University of San Luis Potosí, which was isolated from a mine in Durango, Mexico. The medium contained the following ingredients (g/L): 0.2 - ammonium sulfate, 0.5 - magnesium sulfate, 0.25 - calcium chloride, 3 - dipotassium hydrogen phosphate and 0.005 - iron sulfate. The agar medium was made with the same concentrations, only 12.5 g/L was added. Both mediums were adjusted to a pH of 4.0 and autoclaved at 121 Celsius for 15 min.
We did electroporation, DNA was prepared with the 2013 Igem kit Plate 5 “1G”, it contained pSB1A3 with Amp resistance. We used the strains of Top10, BL21, Shuffle and DH5-Alpha.
We made calcium-competent cells of the strains Shuffle, BL21, Top10 and DH5-alpha. A first experiment was carried to evaluate the growth of A.Thiooxidans in liquid medium with elemental sulfur and sodium thiosulfate. Group 1 had 10 g/L of elemental sulfur, group 2 had 10 g/L of sodium thiosulfate and group 3 had 5 g/L of elemental sulfur + 5 g/L of sodium thiosulfate. The groups of the experiment consisted in: (1) Control medium without bacteria, (2) Control medium with bacteria without genetic modifications, (3) Bacteria with tetH overexpression. It was expected that the curve of the group 3 had the biggest slope value
In this day we made heat shock transformation with the calcium-competent cells of the previous day. The parts of iGEM that were chosen was
The result of the heat shock were analyzed. The antibiotics worked, there was no transformation and the C.C of BL21 were dead.
The strain of Top-10 was the only one that grow, so it was left to grow another night at 37 degrees C and 250rpm. A miniprep was made of the Top10 Cam cells) and was left in the fridge so we could make electrophoresis another day. A transformation was made of the strains BL21, DH5-Alpha and Top10 with RFP Coding Device and pSB1C3.
Digestion and Electrophoresis Gel was made. For the digestions we used XbaI and PstI. We used the latter “Quick- Load Purple 2-Log DNA Ladder.”
Two minipreps was made of Top10 with culture media with CAM with RFP Coding Device.
Transformations were made with the calcium-competents of the day July 21th. The DNA is a RFP coding device of the miniprep of July 26th.
Transformations were made again by heat-shock with the C.C of July 27th.
A transformation (heat-shock) was made with with the strains of July 21: Shuffle, BL21, DH5-Alpha and Top10. Digestion were made using the miniprep of July 26.
Digestions were made of the miniprep of July 26h (0) and the minipreps of July 28th (1, 2). EcoRI, PstI and XbaI were used. Afterwards, an electrophoresis gel was made with Diamond Dye and the Digestions.
A purification of gel was made thanks to a protocol that a collaborator gave us. Calcium-Competent cells were made of the strain DH5-Alpha. Later on, a transformation by heat-shock was made of the same C.C cells.
An inoculum of BL21 was left to grow to do in the following days electrocompetent cells.
An electroporation transformation was made with the iGEM kit plate 3 of 2014, 20L.
Transformation by heat-shock. It was used the promoter AraC/pBad of the iGEM kit 2015 plate 5 17N(BBa_I0500). Also, a GFP was taken out of the iGEM kit plate 1 2015, 14F(BBa_K577881). The strain that was used is C.C DH5-alpha of the day August 4th.
An inoculum of Violaceum was left growing overnight. A transformation of E.coli was made by heat-shock using the iGEM kit parts of 2016 plate 1: 14F(GFP) and Plate 5 17N (Promoter)
An inoculum of Violaceum was left in petri-dishes for it to grow at different dilutions: 1, 1:100, 1:1000. Also, an inoculum was left with Thiobacillus.Unfortunately three weeks after our first inoculum we lost our strain due to a failure in the pH measurements, when we tried to inoculate them again to fresh media cells would not grow. It was found that the pH of the original strain had come down to a pH of 0.63, thus all organic material was dissolved.
Dilutions of violaceum were made again in petri dishes. 8 petri dishes were made, 2 with AMP, 2 with KN, 2 with CAM and 2 controls one of each with a dilution of 1:1000 and another one complete.
A control of Thiooxidans was prepared to have a reference in Abs and solubility. A transformation was made in the strain DH5-alpha by heat-shock. The DNA that was used was from the iGEM plate kit 2016 plate 1: 14F, plate 5:17N. the antibiotic that was used is chloramphenicol.
Test of validation of C. violaceum. To prove we had the correct bacteria we expose a little sample to peroxide and the Gram stain.
A miniprep was made out of the transformations that were made in August 16th.
A digestion was made out of the miniprep of the previous day, with EcoRI and PstI. This digestion didn’t worked correctly because of the backbone and the DNA was almost the same size, so another digestion was made this time with a cut in the backbone with SaoI. Afterwards an electrophoresis gel was made. Also, a purification of the gel was made following the protocol of PureLink Quick Gel Extraction Kit.
Digestions and ligations were made. First the ligation of the backbone pSB1K3 with the iGEM part 14F Plate 1 2016 BBa_K577881. The purification was used of the previous day and the plasmids that were used were the ones of the iGEM kit plates pSB1K3.m1 of 2015 and 2016. Also, a transformation was made with DH5-Alpha with the ligations 14N+ PSB1K3 (years 2015, 2016). A culture media for Thiooxidans was prepared.
A culture media LB was made with CuCL2 * 2H2O to see if Violaceum could become purple or to see if it could make copper precipitate. 4 solutions were made. A transformation was made with LuxI and DH5-alpha. LuxI= BBa_K805016, 19P.
Testes of CuCL2*2H2O in E.coli and C. violaceum. Also, a transformation was made with DH5-alpha with ligations of 14F+pSB1K3 via heat-shock.
A miniprep was made of the transformations of August 22. A transformation was made later on by heat-shock of E.coli DH5-Alpha with the iGEM part 17N(part I0500) of the plate of 2015 and 2016. (Two tests).
Electrocompetent cells were made of C. Violaceum.
6 samples of different mixes were left with “agua regia” for 14 hours.
There is no thiooxidans due to the low pH of 0.59 by the trials of “agua regia”. Digestions were made of the 14F with PstI, EcoRI and SacI.
An electrophoresis gel was made of the digestions of August 27th.
A transformation was made with BL21-Star and DH5-alpha to prove viability. It was took the miniprep RFP+CAM of july 28th.
An analysis of the transformations was made. Also a transformation was made by heat-shock of the constructs: Inducible promoter, cyanide hydratase, sulfur reductase, iron oxidase, control without antibiotic and control with antibiotic.
An inoculum of culture media and antibiotic was made with the following:
DH5-Alpha- pAra + GFP -CAM
DH5-Alpha- pAra- CAM
BL21-Inducible Promoter- AMP
BL21-Cyanide hydratase- AMP
BL21-Sulfur reductase- AMP
BL21-Iron oxidase- AMP
DH5-alpha- pAra +GFP + pSB1K3- Kn
DH5- pAra +GFP +pSBUK3- Kn
Another tube with Violaceum arrived to our laboratory. Also, minipreps, digestions and electrophoresis gel was made out of the 6 parts.
A transformation was made of the parts to test pH (19J and 11P) that have a weak promoter Anderson. An inoculum of Violaceum was left and also an inoculum of 6 constructs to repeat miniprep and gel.
Transformations were made of the parts 14S and 11P of the plate of 2015. In here we stop trying with Thiooxidans, due to it’s lack of growment.
Digestions were made of the following promoters:
Inducible promoter
Cyanide Hydratase
Sulfur
Iron
pAra
pAra + GFP
An electrophoresis gel was made out of the digestions.
Protein extraction of iron oxidase and sulfur oxidase. Also, transformations were made out of the iGEM kit plate of 2015 19J and 11P by heat-shock.
Six transformations were made:
BBa_J23100 (Anderson 1) 2015 Plate 4, 17D. Resistance: Amp
BBa_ J23106 (Anderson 0.47) 2016 Plate 4, 17P. Resistance: Amp
BBa_J23114(Anderson 0.1) 2016, Plate 4, 19J. Resistance: Amp
BBa_KI350005(Resistance pH- proton pump) 2016, Plate 5, 11P. Resistance: Chloramphenicol
BBa_K873002( HSP promoter) 2015, Plate 1, 3H. Resistance: Chloramphenicol.
(RFP), 2015, Plate 3, 11N. Resistance: Chloramphenicol.
Also, digestions were made of Iron Oxidase and Sulfur reductase. Afterwards, an electrophoresis gel was made with this digestions with the 2-log ladder.
Minipreps were made of HSP and RFP. Transformation was made of the cells that didn’t work on september 10 by heat-shock.
Minipreps were made of: 1) Inducible promoter. 2) Iron Oxidase. 3) Sulfur reductase.
Double digestion was made out of the plasmids iGEM gave us with RFP.
Digestion and Electrophoresis was made. Also, 5 transformations with 3 controls.
Cyanide hydratase.
Sulfur reductase
Iron oxidase
Inducible promoter
HCN
1. Control BL21
Control with culture media
Control HCN without AMP
A culture was made with the transformations of September 16 and were left growing to make miniprep.
A miniprep was made out of the 5 parts of september 16.
Also, transformations were made of the promoter BBa_K608003 (2014) by heat-shock.
Transformation was made out of the following parts out of the 2015 Plate 1. Everything was transformed with the C.C BL21:
5A- BBa_Kl8003
5C- BBa_608004
5E- BBa_608006
5G-BBa_ 608007
17B- BBa_K864100
A transformation was made of BL21 of the C.C.
7 Digestions were made, 5 of the lasts parts and 2 of minipreps that were made in July. Also, minipreps were made of the transformations of September 19. An electrophoresis gel was made with EtBr out of this digestions. An inoculum was left out growing to do Calcium-competent cells.
Calcium-competent cells were made
