iGEM 2016 — Team Tec-Monterrey

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# Team:Tec-Monterrey/Notebook

iGEM 2016 - Tec de Monterrey

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# July

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 26 | 27 | 28 | 29 | 30 | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 | 1 | 2 | 3 | 4 | 5 | 5 |

# August

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 30 | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |

# September

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 28 | 29 | 30 | 31 | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |

# October

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 25 | 26 | 27 | 28 | 29 | 30 | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 | 1 | 2 | 3 | 4 | 5 |

# 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

# Tuesday, September 27, 2016

# Friday, September 30, 2016

# Saturday, October 01, 2016

# Sunday, October 02, 2016

# Monday, October 03, 2016

# Tuesday, October 04, 2016

# Wednesday, October 05, 2016

# Thursday, October 06, 2016

# Friday, October 07, 2016

# Saturday, October 08, 2016

# Sunday, October 09, 2016

# Monday, October 10, 2016

# Tuesday, October 11, 2016

# Wednesday, October 12, 2016

# Thursday, October 13, 2016

# Friday, October 14, 2016

# Saturday, October 15, 2016

# Sunday, October 16, 2016

# Monday, October 17, 2016

# Wednesday, October 19, 2016

### We tested our antibiotics: ampicillin, kanamycin, chloramphenicol with our PLYS and Shuffle bacteria

### 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

### We took out from the iGEM plates the following parts: 20F from 2015 plate and 13K from 2012 plate.

### An inoculum of C. violaceum was left to make electrocompetent cells. Also, a test of antibiotic resistance was made in C. violaceum.

### OD was measured 3 times in different times of the inoculum (every 45 minutes). Also, a miniprep was made trying a new kit.

### Digestions were made of the miniprep of the previous day. A growth kinetics curve was made from 9:20 to 18:50. Also we made electroporation of C. violaceum with RFP.

### We made electroporation of C. violaceum. Also, transformations were made of RFP (20F) in E. coli culture media LB+CAM.

### Inoculums were left to grow of the following: Inducible promoter GolS Proton Pump Sulfur Reductase Iron Oxidase Cyanide Hydratase Inverter G+R+T Also, a purification was made of an electrophoresis gel of the fragments that contained pSB1C3. Inoculums were left of the promoter PR5 and Inverter. Digestions were made of Sulfur reductase, GolS and Cyanide hydratase, afterwards an electrophoresis gel of these digestions.

### Transformations were made of the following: mRFP+2T= 20F Inverter Inducible Promoter PR5 Proton Pump GolS HCN Sulfur Reductase Iron Oxidase Cyanide Hydratase Minipreps were made of PR5, Inverter, Proton Pump. Digestions were made of these Minipreps. A purification was made of the electrophoresis gel that was made the day before of GolS, Cyanide Hydratase and Sulfur reductase. Ligation of Sulfure reductase + pSB1C3+ GolS and Cyanide hydratase+pSB1C3 Calcium competent C. violaceum were made based on the following spectrophotometric readings: OD: 0.400 \*\* OD: 0.500 \* OD: 0.600 \*\*

### Transformations were made of the part BBa\_E0840 (RBS+GFP+2T). Digestions were made from the following minipreps: PR5, Proton Pump, Inverter, and an TFP coding sequence for its backbone. For later testing of the Proton Pump, the pH of LB medium was changed into 15 different tubes, into groups of 3 with pH 7, 8, 9, 10 and 11. An electrophoresis Gel was run with today’s digestions, but the PR5 didn’t showed the desired results. Then, the parts were purified using a Aligen purification kit. Afterwards, ligations of these parts was made, joining the proton pump with the backbone (psb1c3) with a 1:1 vector insert ratio. Transformation of C. violaceum was made with RFP using Heat Shock (60 and 80 seconds) 0.1 μL of DNA and 50 μL of CaCl2 were used. OD 0.500 cells and 60 seconds Heat Shock produced the largest number of transformed colonies.

### Transformations of 3 different promoters and an RFP without promoter were made: 5C K608004 (PR2) Promoter 5E K608006 (PR5) 3O K608002 20 F (RFP) Digestions were also made of the Inducible promoter and the PR5. Transformation of the ligations of G,S, C also of the PR5 promoter and the 4 parts previously stated. Gel extraction of the Heat Shock Promoter. Calcium competent cells were made of BL21.

### Transformations of the BL21 cells were made with Gol S (G), Cyanide hydratase(C) and Sulfur reductase(S), all with resistance to kanamycin.

### The following DNA was gathered from the iGEM plate 3 of 2014: BBa\_J06702 → CAMr. RBS + RFP + 2T (280 bp). Total size: 2940 bp. BBa\_K608003 → CAMr. Promoter + RBS (56 bp). Total size: 2100 bp. BBa\_K608006 → CAMr. Promotor + RBS (56 bp). Total size: 2100 Transformation of BL21 calcium competent cells from October 7 using the same conditions as the ones used on October 8, with BBa\_J06702 (1), BBa\_K608003 (2), BBa\_K608006 (3), and RFP (4).

### Single colonies were isolated from cultures (1) and (4) from October 9 and inoculated with kanamycin at 6:50 pm. Calcium competent BL21 cells were transformed via Heat Shock (60 seconds), using 1μL DNA: Part K516030 (Kit plate 1\_2015, 9I) → mRFP protein generator (w/o promotor) Part K823006 (Kit plate 2\_2015, 20M) → Anderson promoter S23102 Part K823008 (Kit plate 1\_2015, 22A) → Anderson promoter S23106 Minipreps were made of PR2 and PR5 with K608004/K608006, promotor and RBS. Once digestions were made with SP, 20 μL final volume, an electrophoresis gel was run using said digestion products.

### C. violaceum transformed with RFP and resistance to kanamycin was cultured on solid medium. A stock of C. violaceum + RFP was made for cryopreservation at -80°C with 20% glycerol. A stock of E. coli BL21 transformed with GolS and resistance to ampicilyn was made for cryopreservation at -80°C with 20% glycerol.

### Minipreps were made of the followings: RBS+RFP 9I. RBS+RFP+2T PR2 RFP P22A P20M PR5 GolS Digestions were made of this minipreps. Also, we measured the pH of the bacteria transformed with GolS and Proton pump.

### Digestions were made again of the followings minipreps: P20M RBS+ RFP PR5 Inducible Promoter Inverter GolS RFP An inoculum of TetH was left at night. An electrophoresis gel was made of the previous digestions. As well as the measurements of the Proton pump.

### Miniprep and digestion of the tetH was made. An electrophoresis gel was made for it. Electroporations of the electro- competent cells were made for GolS.

### Miniprep was made of the following: RFP without Promoter RFP without promoter Iron oxidase Inducible Promoter Digestions were made of the following: Iron oxidase Sulfur reductase Proton Pump RFP Coding Device We made an electrophoresis gel of these digestions.

### We did trials with IPTG with the OD of the bacteria with RFP. Ligations were made as: pSB1C3+Protons Pump Inducible P.+RFP1 Inducible P.+RFP2 Digestions were made again of the followings: Iron Oxidase Sulfur Reductase Proton’s Pump Ligations of GolS+pSB1C3 Promoter RFP Electrophoresis was made of these digestions as well as purification. Electroporation of E. coli with the DNA of the ligations: pSB1C3+Protons Bomb Inducible Promoter+ RFP Inducible Promoter+RFP Inducible Promoter + Inverter Digestions were made as well as the electrophoresis gel. A purification was made of: tetH, pSB1C3, RFP and HSP.

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