Team:Sheffield/Project/lab
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+ | 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 | Friday 9th July: First Day in the Labs | ||
- | Looked through the Freezers to see what was left by the previous 2008 iGEM team. | + | 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. | + | 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]] |
Latest revision as of 00:45, 28 October 2010