Team:UCC Ireland/Notebook
Week 1
Monday
Interlab plate reader protocol
Transformations:
- 14B plate 1: TetR
- 14D plate 1: TetR
- Interlab test d.1
- Interlab test d.3
- Interlab test d.4
- Interlab neg. control
- Interlab pos. control
- Internal d.2
Cell Measurement Protocol:
- Interlab test d.2
- Interlab test d.5
- Interlab test d.6
- Interlab pos. control
Inoculated cultures into LB and put on shaker for 16 hours
Editing the Wiki page
Tuesday
Interlab experiment continued: cell growth sampling assay on various samples.
Brainstorming project name and logo
Wednesday
Meeting at 2pm and presentations
Interlab cell measurements
Thursday
Finished interlab study
Safety form completed
Tet plasmids examined
LB, construct, Tet
Friday
BBa_K577893 and BBa_K577895
TetR repressed GFP and RFP plasmids respectively were grown in overnights in concentrations of tetracycline antibiotic of:
For GFP
- 2.5mg/L 19831 20815
- 0.5mg/L 43730 44945
- 0.25mg/L 46470 42639
- 0.05mg/L 46395 43938
- 0.025mg/L 45420 46352
- 0.005mg/L 39398 40997
- 0mg/L (Control) 30017 31865
New Concentrations:
- 0.10mg/L
- 0.20mg/L
Week 2: 3rd of July
Monday
Transformation of:
- BBa_c0040 - Plasmid with TetR gene
- BBa_I13600 - Plasmid with cfp reporter
- BBa_J23119 - Plasmid with constitutive promoter
- BBa_K1357008 - Plasmid with purple fluorescent protein
- BBa_K1357009 - Plasmid with amilCP gene
3A assembly method
Standard assembly method
Tuesday
Overnight cultures of the transformations
Wednesday
Meeting with Dr. Paul Young
Thursday
- Miniprep of TetR plasmid.
- Restriction digest using EcoR1 and Pst1.
- Control without enzymes
^Ladder. Restriction digest. Plasmid only.
Week 3: 10th of July
Monday
Mini digest of plasmids using EcoR1 and Pst1:
- BBa_K1357008 (purple)
- BBa_K1357009 (amilCP)
- BBa_l13600 (cfp)
Ran these samples on gel
Correct bands obtained.
3A Assembly protocol begun - Desired results:
- Purple+cfp
- AmilCP+purple
- Negative control (just backbone)
Began by carrying out a restriction digest on the plasmids using the 3A assembly
Transformation of:
- BBa_K592009 (AmilCP blue)
- BBa_E1010 (AmilCP red)
- BBa_K592011 (AmilCP green)
- BBa_K145279 (GFP TetR)
- BBa_K592012 (AmilCP pink)
Tuesday
No coloured colonies on plates yet.
3A Assembly continued:
Ran a gel in order to check that plasmids had been correctly cut for our 3A Assembly protocol.
Marker, Backbone, Backbone, Backbone, AmilCP+EcoR1+Spe1, Purple+Xbal+Pst1, Cfp+EcoR1+Spe1, Purple+Xbal+Pst1.
Correct bands obtained.
Performed a ligation reaction and transformed the ligated plasmid into E.Coli.
Wednesday
Plasmid preps of AmilCP plasmids (coloured plasmids).
3A assembly results:
- Negative control grew
- One/Two colonies grew in 3A assembly plates
- Enzymes did cut as shown from gel.
Meeting with IGNITE - Business plan
Meeting with Dr. Paul Young -2pm
- Need to clone promoter genes using 3A assembly with reporter genes (AmilCP)
- GFP+TetR plasmid - test with concentrations of tetracycline (BBa_K145279
- BBa_K577895 (TetR and RFP plasmid)
Thursday
- Plasmid preparation of ligated plasmid (cap reported+purple reported+amp backbone) from overnight liquid culture
- Restriction digest on the following plasmids to check correct plasmids are obtained. (This test is needed as colonies did not produce any colour on plate. No promoter was present in plasmid to produce colour):
- 1. Ligated Amp. backbone + BBa_I13600 (cap reporter) + BBa_K1357009 (purple reporter)
- 2. BBa_K592009 (AmilCP blue)
- 3. BBa_E1010 (AmilCP red)
- 4. BBa_K592011 (AmilCP green)
- 5. BBa_K52012 (AmilCP pink)
- Ran a gel with these samples - both uncut and cut.
Week 4: 17th of July
Monday
Plasmid preps of:
- BBa_K592012 (AmilCP pink)
- BBa_K592011 (AmilCP green)
- BBa_K1357009 (amilCP blue)
- BBa_E1010 (AmilCP red)
- BBa_J23119 (Promoter)
3A assembly protocol began:
- Restriction digest of promoter, AmilCP pink, AmilCP blue.
- Ligation of promoter + gene + backbone.
Transformation of plasmids containing promoters:
- BBa_I14033 - P(Cat)
- BBa_J23100 - Constitutive promoter
- BBa_J23101 - Constitutive promoter
- BBa_J23117 - Constitutive promoter
Tuesday
Plasmid preps of the following promoters:
- BBa_J23119 x2
- BBa_K592012 (AmilCP pink)
- BBa_K592011 (AmilCP green)
- BBa_K1357009 (amilCP blue)
- BBa_E1010 (AmilCP red)
Used the nanodrop to quantify the purity and amount of DNA in the samples
Chose BBa_592012 and BBa_E1010 to digest along with the promoter.
Ligation reaction of BBa_592012 with the promoter in the Amp backbone
Ligation reaction of the BBa_E1010 with the promoter in the Amp backbone
Transformation of the ligated plasmids onto Amp plates.
Fom previous day: 3A assembly continued:
- Transformation of the frozen ligated plasmids, and plated onto Amp plates.
Colonies present on pCat plate: Overnights made from these cultures.
0.8% w/v agarose gel ran of uncut BBa_J23119 vs cut BBa_J23119. Against a 1 kb ladder. 5µl of the ladder was loaded into the first well and 15µl of each sample of the 2nd and third wells.
- Plasmid DNA was retrieved from the distribution kit and mini-prepped. The concentration of the plasmid DNA was checked using a nano-drop apparatus and was detected as 22ng/µl of nucleic acids.
- A restriction digest was carried out using Ecor1 and Pst1 to cut the plasmid seen below into fragments of 77BP and 2,028BP, which can be seen visualised on the gel to the right versus the uncut plasmid.
Wednesday
Colonies on 3A assembly plates except the colonies are colourless!!?? - No ribosome binding site.
Overnights made from the colourless colonies - To be tested on a gel.
Plasmid preps of pCat promoter and tested on nanodrop. Restriction digest on the sample.
Transformation of the following:
- BBa_J06702 - mCherry, Plate 3, 15B
- BBa_K592025, Plate 1, 19C
- BBa_I13502
Thursday
Overnights made of the transformed colonies from Wednesday.
Plasmid prep of E1010 and K592012, nanodrop analysis;
- E1010 - 47ng of Nucleic acids/ml
- K592012 - 48ng of Nucleic acids/ml
- K592012 - 45.7ng of Nucleic acids/ml
- K592009 - 46.2ng of Nucleic acids/ml
Cut all of the plasmid preps using Ecor1 and Pst1 and ran on a 0.8% agarose gel to check that the correct recombinant plasmid had been taken up by the competent cells. Predicted fragment sizes are as follows:
- E1010 - 741BP and 2,155BP
- K592012 - 716BP and 2,155BP
- K592009 - 704BP and 2,155BP
Strange results obtained. Possible reasons for fragments of approximately 2000bp seen:
One of the restriction enzymes, EcoR1 or PstI, did not cut the ligated plasmid since the restriction sites might have been destroyed during ligation
Friday
Plasmid prep of yesterday’s overnights: BBa_J06702 - mCherry, BBa_K592025 and BBa_I13502. Digested these plasmids with Xba and Pst.
Promoters (PCat and J23119) digested with EcoR1 and Spe.
Backbones (tet) digested with EcoR1 and Pst1.
Met Yensi, shown how to use BMG fluostar omega plate reader in CCRC.
Ligation:
Equation: ng insert = vector ng x (kb size insert/kb size vector) x molar ratio (3) Carried out a ligation reaction.
Made overnights of TetR, GFP-TetR, Promoter-RFP, Promoter-GFP.
Week 5: 24th of July
Monday
Ligations from last week worked!
- AmilCP blue colonies present
- mCherry red colonies present
- No ligase control of AmilCP
Ran repeat of K145279 experiment:
- GFP under control of pTet and TetR.
- OD600 - 0.799 @ T=0 of Cells.
- Ran same six concentrations 2.5µg/L → 0.005µg/L verses blank LB versus a constitutively expressed GFP (BBa_K84001). Also included was a negative control of cells that did not express GFP so we used an overnight of cells for (BBa_K145201).
Experiment:
Ligation of TetR with Promoter-RFP using the Standard Assembly method. This vector will be tested with various concentrations of tetracycline and its fluorescence measured.
-
Plasmid prep of the samples:
- TetR (BBa_K145201) : 113ng/microL
- Promoter.RFP (BBa_I13521) : 50ng/microL
-
Restriction digest:
- TetR with EcoR1, Spe1
- Pro.RFP with EcoR1, Xba1
- Gel extraction of TetR gene and gel digestion.
- Measured the TetR gene concentration. Low yield of 4.3ng/microL obtained. Sample not workable and quite contaminated. Gel extraction will be repeated tomorrow.
Tuesday
Experiment from Monday repeated - Restriction digest of TetR and ran on a gel and excised. Yield of DNA and purity measured using the nanodrop. - Low yield of 6.6ng/microL.
Ligation of TetR + Pro-RFP. Transformation of the ligated plasmid.
Plasmid preps of the overnights : AmilCP and mCherry.
Wednesday
-Gel extraction of TetR gene - try to increase purity and yield. Methods to increase yield of DNA from gel extraction:
- Wash DNA with ethanol multiple times - to remove salt residues
- Make sure all ethanol is gone from DNA - run a gel check on the gel extracted DNA. If the sample floats out of the well you have ethanol contamination.
- Elute with hot buffer (70°C) and allow the column to incubate with the hot buffer for 5 minutes prior centrifugation
- Renature the DNA - If the eluted DNA appears to be half the expected size then it is single stranded. Renature DNA by heating it to 95 degrees for a minute and let it cool slowly to room temperature.
- Run controls - Digest empty vector cut with a single enzyme, perform the gel extraction, and re-ligate it. A vector cut with one enzyme should re-ligate very easily and provide plenty of colonies on the plate. If it does, then the inability to clone the DNA may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA cloned codes for a protein that may be toxic in bacteria.
- Minimize exposure to UV light
- Trim the gel slice as much as possible
-Ligated cultures grew with no RFP signal - TetR repressor protein is preventing the production of RFP. Overnights were made of this cultures.
Thursday
-Church plasmid has arrived - Erythromycin tests begin Make plates using the Church plasmid - streak using pipette tip or inoculating loop
-Experiment with TetR- Pro.RFP : Plasmid prep, restriction digest and run on a gel to confirm parts ligated into plasmid.
-Lanes from left to right - Marker, TetR-Pro.RFP plasmid cut with EcoR1 and Pst, LuxS (Ellen’s experiment). Results are a bit unusual but it seems that the ligation worked.
-Tests using various concentrations of tetracycline to show an increase of fluorescence when tet is added to the system.
-Streaked erythromycin plasmid backbones onto ampicillin plates (x2)
Friday
-Construct primers for G-blocks
-Repeat Tet experiment with the plate-reader.
Week 7: 7th of August
Monday
Ligations from last week worked!
- AmilCP blue colonies present
- mCherry red colonies present
- No ligase control of AmilCP
Ran repeat of K145279 experiment:
- GFP under control of pTet and TetR.
- OD600 - 0.799 @ T=0 of Cells.
- Ran same six concentrations 2.5µg/L → 0.005µg/L verses blank LB versus a constitutively expressed GFP (BBa_K84001). Also included was a negative control of cells that did not express GFP so we used an overnight of cells for (BBa_K145201).
Experiment:
Ligation of TetR with Promoter-RFP using the Standard Assembly method. This vector will be tested with various concentrations of tetracycline and its fluorescence measured.
-
Plasmid prep of the samples:
- TetR (BBa_K145201) : 113ng/microL
- Promoter.RFP (BBa_I13521) : 50ng/microL
-
Restriction digest:
- TetR with EcoR1, Spe1
- Pro.RFP with EcoR1, Xba1
- Gel extraction of TetR gene and gel digestion.
- Measured the TetR gene concentration. Low yield of 4.3ng/microL obtained. Sample not workable and quite contaminated. Gel extraction will be repeated tomorrow.
Tuesday
Experiment from Monday repeated - Restriction digest of TetR and ran on a gel and excised. Yield of DNA and purity measured using the nanodrop. - Low yield of 6.6ng/microL.
Ligation of TetR + Pro-RFP. Transformation of the ligated plasmid.
Plasmid preps of the overnights : AmilCP and mCherry.
Wednesday
-Gel extraction of TetR gene - try to increase purity and yield. Methods to increase yield of DNA from gel extraction:
- Wash DNA with ethanol multiple times - to remove salt residues
- Make sure all ethanol is gone from DNA - run a gel check on the gel extracted DNA. If the sample floats out of the well you have ethanol contamination.
- Elute with hot buffer (70°C) and allow the column to incubate with the hot buffer for 5 minutes prior centrifugation
- Renature the DNA - If the eluted DNA appears to be half the expected size then it is single stranded. Renature DNA by heating it to 95 degrees for a minute and let it cool slowly to room temperature.
- Run controls - Digest empty vector cut with a single enzyme, perform the gel extraction, and re-ligate it. A vector cut with one enzyme should re-ligate very easily and provide plenty of colonies on the plate. If it does, then the inability to clone the DNA may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA cloned codes for a protein that may be toxic in bacteria.
- Minimize exposure to UV light
- Trim the gel slice as much as possible
-Ligated cultures grew with no RFP signal - TetR repressor protein is preventing the production of RFP. Overnights were made of this cultures.
Thursday
-Church plasmid has arrived - Erythromycin tests begin Make plates using the Church plasmid - streak using pipette tip or inoculating loop
-Experiment with TetR- Pro.RFP : Plasmid prep, restriction digest and run on a gel to confirm parts ligated into plasmid.
-Lanes from left to right - Marker, TetR-Pro.RFP plasmid cut with EcoR1 and Pst, LuxS (Ellen’s experiment). Results are a bit unusual but it seems that the ligation worked.
-Tests using various concentrations of tetracycline to show an increase of fluorescence when tet is added to the system.
-Streaked erythromycin plasmid backbones onto ampicillin plates (x2)
Friday
-Construct primers for G-blocks
-Repeat Tet experiment with the plate-reader.
Week 8: 14th of August
Monday
- Resuspended primers
- Diluted primers down to a concentration of 10microM
- Made up fresh M9, LB agar and LB broth.
- Made fresh Cam and Amp plates.
- Plated more Church plasmid cultures and ligated Tet cultures.
- PCRed up Erythromycin, Mdh and FrmR g-blocks with primers to add correct adapters.
- Ran a gel to confirm that the PCR worked successfully.
- Received PRSF-duet backbone.
- Visited Blackwater gin microdistillery in Waterford
Tuesday
- Restriction digestion of the Prsf-duet backbone with Acl1 enzyme.
- Ran a gel to confirm the digestion worked
- New primers designed and ordered.
Wednesday
- Set up the growth curve experiment using ligated TetR-RFP and Church plasmid. Put in plate reader overnight shaking.
- Ran the PCR experiment of the gblocks a final time - Strange results?? - Confirmed that primers are incorrect.
- Made Tetracycline plates
Thursday
- Growth curve experiment examined. Here are the results:
- Experiment testing the OD of the amilCP cells at T0, T1.5, T3, T4.5, T6. Cells left shaking in a flask. 1mL taken from the flask at the various time points. From this, 300microL mixed with 2.7mL of m9 and OD measured. 10^-1, 10^-2, 10^-3 dilutions made taking from the 1mL and plated. Colonies will be counted tomorrow and number correlated with the dilution.
Friday
- PCRed up the iGEM linearized backbones
- Ran these samples on a gel to confirm the PCR worked
- Transformed two plasmids into DH5A E.coli via heat-shock : GFP producing plasmid and a plasmid containing a constitutive promoter. These plasmids will be ligated next week to form a constitutively producing GFP plasmid.
Week 9: 21st of August
Monday
- Made overnights in M9 and LB of AmilCP, Church plasmid, and in m9 controls (Cells only, promoter J23119 plasmid, and Yensi’s GFP inducible control).
- Poured Tetracycline plates and completed making new M9 media
- Decided on a protocol for Tuesday’s experiments and laid out hood
Tuesday
Experiment to measure the Church GFP fluorescence vs the Erythromycin concentration
- Measured the OD’s of the overnights. The OD’s were as follows:
- Refreshed the cells in new M9 media and adjusted their OD’s to 0.05. Placed in a 50mL tube and left shaking for 3hrs.
- After 3 hrs the OD’s were as follows:
- Got a clear bottomed dark plate and pipetted the respective cells into each well. Added the correct concentration of Erythromycin to each well.
Fig. 1. Graph showing average fluorescence of Negative control (Cam resistant bacteria expressing promoter), 0.04µg/mL Erythromycin (Legal limit concentration) and 40µg/mL Erythromycin (Highest concentration used. Average fluorescence is average fluorescence over three replicates minus blank.
Fig. 2. Graph showing all average results. As seen, IPTG induced GFP strain is unsuitable as a positive control due to expression levels far above that of other strains used, drowning out their signals.
Fig. 3. Graph showing fluorescence response to all Erythromycin concentrations minus the positive control.
Experiment was carried out with a lid on the 96 well plate to prevent evaporation of the samples. It was found afterwards that the lid is a significant source of autofluorescence. As seen from the graphs, the negative control has a higher RFU than all of the samples excluding the highest concentration of Erythromycin used (40µg/mL). This was possibly due to the lid.
Wednesday
- Refreshed overnights (AmilCP M9 1%, Cells only, promoter J23119) and made to an OD of 0.05.
- Began AmilCP test on the plate reader. This test will examine the rate of AmilCP production vs the OD of the cells.
- Discovered that the lid of the plate reader fluoresces in the plate reader.. This causes an error in our GFP results.
- Made overnights of TetR and RFP. These constructs will be used to make a new ligated TetR-RFP construct to use as our tetracycline biosensor.
Thursday
-
Ran a gel on the pure resuspended gblocks to confirm that there is no contamination between gblocks.
It seems like there is some sort of contamination in the pure gblocks or the primers are binding and amplifying other sites??
A gel extraction may have to be carried out to get the correct gblocks.
- 3A Assembly of TetR-RFP plasmid:
- Plasmid prep of the TetR and RFP respectively. Measured conc. And purity on nanodrop.
- Restriction digest of the fragments using the 3A restriction enzymes
- Ran the digest on a gel to confirm the digest worked:
- Lanes: Ladder, cut TetR, uncut TetR, cut RFP, uncut RFP, cut backbone, uncut backbone.
- 3A assembly ligation run for 50 minutes with T4 Ligase enzyme.
- Transformed cells with the new ligated TetR-RFP plasmid.
Friday
