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{{Template:Team:Sheffield/Navigation2}} This lab book contains a day to day account of what happened in the lab and processes we took into creating our bio-bricks and bio-sensor. It also contains many of the protocols we have used and results such as pictures from gel electrophoresis. Friday 9th July: First Day in the Labs Looked through the Freezers to see what was left by the previous 2008 iGEM team. Found two strains of E. coli, DH5 alpha and MG1655, which were spread onto LB agar to test for viability. Monday 12th July: E.coli are Viable Both strains grew showing that they are viable. Overnight primary cultures of each strain were set up in 5ml of LB broth Tuesday 13th July: Practice Transformations and Competency Experiments Both strains of E.coli were made chemically competent[[1]] using the overnight cultures made on the 12th. The now competent cells were transformed with part BBa_J45014 from the iGEM kit plates(Banana odour).[[2]] This was done to test both our transformation and competency methods. After transformation the cells now containing part BBa_J45014 were spread onto ampicillin agar plates and put into a 37°C incubator overnight. Wednesday 14th July: Transformation Failure Neither strain grew on the Amp plates. Suggesting that either our cells were not competent or that the transformation did not work. Thursday 15th July: BarA Knock Out Arrives BarA Knock out KSB837 strain E.coli arrives. 5ml of LB broth was inoculated with the BarA KO and left to grow in a 37°C incubator overnight. Friday 16th July: Second Attempt at Transformations The transformation attempted on the 13th of July was repeated and left to grow over the weekend.[[1]] BarA KO grew as expected. Monday 19th July: Transformations did not grow. Third attempt at transformation this time using two different banana odours and a winter green odour bio-bricks from the iGEM kit.[[1]] Thursday 22nd July: The Transformations grew. Tested the banana using isoamyl alcohol which produced a banana smell. However the lab did not have the correct substrate for the winter green odour producing cells so we could not confirm that these cells transformed. Friday 23rd July: Made some competent BarA knockout MG1655 E.coli cells and stored them in the freezer with glycerol [[1]]. Monday 26th July: Made overnight starter cultures of E.coli containing pACYC Duet-1. Tuesday 27th July: The control also grew. Made new LB repeated overnight cultures. Wednesday 28th July: Made glycerol stock of MG1655 with pACYC duet-1. Carried out a plasmid mini prep with the remaining samples of MG1655 with pACYC duet-1.[[1]] Checked the plasmid structure using restriction enzymes and then gel electrophoresis [[2]]. Thursday 29th July: Transformed the pACYC duet-1 mini prep with BarA KO strain e.coli.[[1]] Friday 30th July: Transformations failed. Transformations from yesterday repeated. Monday 2nd August: Transformations failed again. Made new LB and double resistance agar plates (Km and Cm). Attempted transformations again.[[1]] Tuesday 3rd August: BarA KO with pCAYC duet-1 transformation worked. Made BarA KO competent cells.[[1]] Made more glycerol stock of MG1655 and DH5alpha. ( to use for controls) Made overnight culture of BarA KO with pACYC duet-1 from the successful transformation. Wednesday 4th August BarA KO with pACYC duet-1 overnight cultures grew and were stored at -20°C Tuesday 10th August: Made overnight cultures of MG1655 WT and pCola. Made more LB and agar plate with Cm. Wednesday 11th August: Carried out a plasmid mini prep to isolate pCOLA which was then stored at -20°C.[[1]] Transformed pSB1C3 plasmid into MG1655 WT.[[2]] Thursday 12th August: Transformations failed. Possibly due to unknown resistance on the pSB1C3 plasmid. Made an overnight starter culture culture with pCDF. Friday 13th August: Carried out a plasmid mini prep to isolate pCDF which was then stored at -20°C.[[1]]. Monday 16th August: Poured more antibiotic agar plates, (Km, Amp, Km + Amp) Set up a restriction digest with pACYC, pCDF and pCOLA with EcoR1 and Pst1 enzymes: Plasmid- 10μL Pst1 - 2μL NcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Carried out gel electrophoresis [[1]] of the digested plasmids. Made overnight starter cultures of MG1655 WT. Made new LB. Tuesday 17th August: Made MG1655 chemically competent.[[1]] Transformed the MG1655 competent cells with several GFPs, E.chromis and LacZs from the iGEM kit. ( Parts BBak274100, BBak274200, BBak274001, BBak274002, BBak274003.)[[2]] Wednesday 18th August: All transformations failed. Made overnight starter cultures of MG1655 WT and DH5 alpha WT. Thursday 19th August: Made MG1655 and DH5 alpha e.coli chemically competent.[[1]] Made more stock antibiotics. Friday 20th August: Transformed the Pga promoter and GFPs (from the iGEM kit) with the chemically competent DH5 alpha cultures.[[1]] Monday 23rd August: Transformation of pga promoter was successful. Transformations of GFPs and e. Chromi failed and repeated. Set up an overnight restriction digest of plasmids pACYC-duet, pCDF and pCOLA using Hind3 and Nco1 restiction enzymes: Plasmid- 10μL Hind3 - 2μL Nco1 - 2μL buffer - 2μL BSA (x20) - 1μL H2O - 3μL Transformations failed. Ordered competent DH5-alpha e.coli from Bioline. Set up an overnight restriction digest of pgaA promoter with EcoR1 and Pst1 restriction enzymes: Plasmid- 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Ran an electrophoresis gel[[1]] of plasmids pACYC duet-1 and pCDF. Results shown bellow: <p> <img src="https://static.igem.org/mediawiki/2010/7/70/2408.JPG" /> </p> The first three wells from the bottom were filled with the cut pACYC duet-1. The three wells between the two ladders contain the cut pCDF. pACYC duet-1 was extracted from the gel.[[2]] Wednesday 25th August: Gel electrophoresis[[1]] was carried out on the digested pgaA promoter but the band was too faint to be extracted. Attempted PCR of the BarA gene from DH5-alpha WT. Gel electrophoresis [[2]] was then carried out on the PCR products. However the results shown below suggests PCR was unsuccessful. <p> <img src="https://static.igem.org/mediawiki/2010/9/92/2508.JPG" /> </p> Thursday 26th August: Attempted PCR of BarA a second time but again results are gel electrophoresis were poor. Repeated gel electrophoresis [[1]] of the cut pgaA promoter which produced better results: <p> <img src="https://static.igem.org/mediawiki/2010/6/69/2608.JPG" /> </p> The now isolated pgaA promoter was extracted from the gel.[[2]] Set up an over night restriction digest of pACYC using Hind3: Plasmid- 10μL Hind3 - 2μL buffer - 2μL BSA (x20) - 1μL H2O - 3μL Tuesday 31st August: Many overnight restriction digests were set up as shown bellow: 1) pACYC Plasmid - 35μL Nco1 - 1μL Buffer 3 - 4μL BSA (x100) - 0.4μL 2) pSB1C3 Plasmid- 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 3) pga Promoter Plasmid - 10μL EcoR1 - 2μL EcoR1 buffer - 2μL Spe1 - 2μL BSA (x20) - 1μL H2O - 3μL 4) csrB Plasmid - 10μL EcoR1 - 2μL EcoR1 buffer - 2μL Pst1 - 2μL BSA (x20) - 1μL H2O - 3μL 5) Part BBa_E0040 (GFP) Plasmid - 10μL EcoR1 - 2μL Spe1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Wednesday 1st September Carried out gel electrophoresis [[1]] and gel extraction[[2]] of the overnight ligations: The gel was set up accordingly, with lane one starting from the top: 1) Ladder. 2) Part BBa_E0040. 3) csrB Promoter. 4) pga Promoter. 5) pSB1C3. 6) pACYC. 7) pACYC. <p> <img src="https://static.igem.org/mediawiki/2010/b/b2/0109.JPG" /> </p> The short bands from lanes 7,6,5,4 and 1 were extracted using the gel extraction kit. Overnight restriction digests were set up as shown below: 1) GFP from lab in unknown plasmid: Plasmid - 10μL Xba1 - 2μL Pst1 - 2μL Buffer 3 - 2μL BSA (x20) - 1μL 2) Part BBa_E0040 (GFP) Plasmid - 10μL Xba1 - 2μL Pst1 - 3μL Buffer 3 - 2μL BSA (x20) - 1μL Thursday 2nd September The overnight digestions were checked using gel electrophoresis [[1]]. The gel was set up as follows starting from the bottom: 1) GFP plasmid from the lab. 2) Part BBa_E0040. 3) Ladder. <p> <img src="https://static.igem.org/mediawiki/2010/b/b5/Gel0209.JPG" /> </p> Both bands in lane 2 were removed using the gel extraction kit.[[2]] * Ligation of pgaA promoter, Part BBa_E0040 and pSB1C3: pgaA promoter - 2μL Part BBa_E0040 - 2μL pSB1C3 - 2μL T4 DNA ligase - 1μL T4 buffer (x10) - 2μL H2O - 11μL Ligation failed as shown by the below gel: <p> <img src="https://static.igem.org/mediawiki/2010/9/9e/0209b.JPG" /> </p> The ligation is in lane one. The three bands show that ligations was not successful. Monday 6th September Multiple overnight restriction digests were set up as shown bellow: 1) Part BBa_E0040: Plasmid - 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 2) Part BBa_E0040: Plasmid - 10μL Xba1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 3) pgaA promoter: Plasmid - 10μL Spe1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 4) CsrB promoter: Plasmid - 10μL Spe1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 5) HapR promoter: Plasmid - 10μL Spe1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL 6) Cholera System: plasmid - 10μL Hind3 - 2μL Nco1 - 2μL Buffer 2 - 2μL H2O - 4μL Tuesday 7th September Carried out gel electrophoresis [[1]] on the overnight restriction digests. Using the gel extraction kit:[[2]] a) Both bands of part BBa_E0040 should have be recovered, but bands in wrong place so not extracted. b)The short promoter bands were recovered. c)The 3800pb band of the system was recovered. Wednesday 8th September Gel Electrophoresis [[1]] was repeated with the two differentlly cut plasmids containing Part BBa_E0040: The Gel was set up as follows starting from the bottom: 1) Part BBa_E0040 cut with EcoR1 and Pst1. 2) Part BBa_E0040 cut with EcoR1 and Xpa1. 3) Ladder. <p> <img src="https://static.igem.org/mediawiki/2010/b/b7/0809.JPG" /> </p> Both bands from each lane were removed using the gel extraction kit.[[2]] A ligation was set up between the cholera system and the cut plasmid pACYC. Six different variations of the ligase constituents were used to see which produced the best results: Substance(μL) Tube 1 Tube 2 Tube 3 Tube 4 pACYC 4 2 2 2 System 2 2 4 2 Ligase 1 1 1 0 Buffer 1 1 1 1 H2O 2 4 2 5 Thursday 9th September The cut promoters were ligated with part BBa_E0040 which has been cut with EcoR1 and Xba1, using the following method for each promoter: Substance (μL) Tube 1 Tube 2 Tube 3 Tube 4 BBa_E0040 1 2 5 2 Promoter 1 5 2 2 Ligase 1 1 1 0 Buffer 1 1 1 1 H2O 6 1 1 5 The different methods were used to see which volumes produced the best results. Friday 10th September Transformed the ligations into chemically competent DH5 alpha cells onto ampicillin agar plates.[[1]] Monday 13th September All transformations were successful. Made overnight starter cultures of the transformations. Tuesday 14th September Carried out plasmid mini preps of all the successful transformations using the overnight cultures from yesterday.[[1]] Set up an overnight digestions of the promoters using the following method: Promoter digest: Plasmid - 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Set up an overnight digestion of part BBa_J04450, using the following method: BBa_J04450 digest: Plasmid - 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Part BBa_J04450 is a RFP contained in a pSB1C3 plasmid and is also Cm resistant. This digestion is therefore to remove the RFP so that this plasmid can be used for submission of our promoter-GFP biobricks to iGEM. Wednesday 15th September The overnight digestions were checked using gel electrophoresis[[1]]. Which was set up as shown bellow: Starting from the top: Lane 1) BBa_J04450 Lane 2) Ladder Lane 3) BBa_J04450 Lane 4) HapR-GFP Lane 5) CsrB-GFP Lane 6) pgaA-GFP Lane 7) CsrB-GFP Lane 8) pgaA-GFP <p> <img src="https://static.igem.org/mediawiki/2010/5/56/1509.JPG" /> </p> The small band from the lanes containing a cut promoter-GFP were extracted using the gel extraction kit.[[2]] The large Band from the lanes containing part BBa_J04450 were extracted using the gel extraction kit.[[3]] Thursday 16th September Ligations were set up between the promoter-GFP and the digested pSB1C3 plasmid (which was retrieved from part BBa_J04450) using the method shown bellow: Substance (μL) Tube 1 Tube 2 Tube 3 Tube 4 pSB1C3 1 2 5 2 Promoter 1 5 2 2 Ligase 1 1 1 0 Buffer 1 1 1 1 H2O 6 1 1 5 These ligations were left for 2 hours then using 5μL they were transformed into chemically competent DH5 alpha cells. Monday 20th September Transformed chimeric plasmid into BarA KO cells.[[1]] Co-transformed the cholera system gene and the HapR-GFP promoter into BarA KO cells.[[2]] Tuesday 21st September Chimeric transformation grew successfully on Amp agar plates. Made overnight primary cultures of the above transformation. Co-transformation grew on successfully on Amp, Cm agar plates. However growth was abnormal so a culture from the centre of the growth was removed and re-streaked. Wednesday 22nd September The re-streak of the co-transformation grew on the Amp, Cm agar plates. Carried out plasmid mini preps of the E.coli containing the Chimeric sequence and of E.coli containing the HapR promoter. [[1]] Overnight restriction digests were set up using the two mini preps of the chimeric sequence and the hapR promoter: Plasmid - 10μL Pst1 - 2μL EcoR1 - 2μL EcoR1 buffer - 2μL BSA (x20) - 1μL H2O - 3μL Thursday 23rd September The overnight digestions were then run on a gel with equivalent undigested parts to check for validity: Lane 1 - Ladder Lane 2 - Cut HapR Lane 3 - Uncut HapR Lane 4 - Cut Chimeric sequence Lane 5 - Uncut Chimeric sequence Friday 24th September Two experiments were set up to test the function of the BarA KO E.coli the contained the cholera system and HapR-GFP promoter in response to the cholera auto-inducer CAI-1: 1.) Simple test: For a simple test of the cholera system based approach the BarA KO strain which contained both the cholera system and the HapR promoter(with GFP) was grown for 1 hour at 37°C in 5ml LB (containing Cm and Amp). Then 5μL of the auto-inducer CAI-1 was added and the E.coli was left to grow again for 2 hours at 37°C. After the 2 hours the sample was centrifuged at 13,000rpm for 1min and excess LB was removed from the top of the sample to concentrate the sample. The concentrated sample was staged on a slide and looked at under a fluorescent microscope which was successfully able to identify bacteria producing GFP. 2.) Characterisation: An overnight flouresomter kinetic was set up for the co-transformed E.coli in a 96 well plate as shown bellow: Row 1 - LB control Row 2 - No CAI-1 Row 3 - 0.5μL CAI-1 Row 4 - 1.0μL CAI-1 Row 5 - 5μL CAI-1 Row 6 - 10μL CAI-1 Row 7 - MG1655 [[Image:Sheffield sponsors.png]]

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