Team:Lethbridge HS/Notebook
Notebook
Record of our work
Please click on the different events on the lab to read more about them. Also, feel free to click on the months on the sidebar to reveal a list of lab dates.
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June
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2015-June-06
Lab book june 6
Names: Jon and Kieran
We stained inoculated biofilms and performed the Crystal Violet assay. Left in 37 C for 36 hours. We did 4 trials 100uL, 4 trials 200uL, and 4 trials 300uL. Biofilm formation was successful, with varying band thickness. We also replicated this with two 5mL LB petri dish inoculated with 20uL E.coli. We added 5mL of crystal violet each to the petri dishes to cover the whole plate.
We also performed an oxalic acid gradient with 0%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 2.0% oxalic acid to give a final volume of 6mL. We added 20uL of E.coli to each 6mL tube. Left in 37 C.
We did a 1/100 dilution of E.coli into LB media, and left it in 37 C for (hopefully) 24 hours. We did 4 trials 100uL, 4 trials 200uL, and 4 trials 300uL.2015-June-08
Lab book june 8
Names: Kieran
Biofilm produced on 24h 96 well plate.
Testing to see if biofilm will produce on 12h 96 well plate (1 in 100 culture dilution).
And also doing 24h 96 well biofilm formation testing to see what detergents and chemical impacts have on the decrease in biofilm production.
*Biofilms placed at 8:00pm
Oxalic acid test run for 12 hours (0, 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.4, 0.5, 2.0%)
*Oxalic acid placed at 7:30pm
Tested the production of parafilm sacs for in vitro Varroa feeding. (using kimwipes and 2mL tubes as containment methods) .2015-June-10
Lab book june 10
Names: Kieran
Performed oxalic acid testing of E.coli growth in LB media at (0, 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.4, 0.5, 2.0%) concentrations. Placed at 6:50pm.
Produced more biofilms.
Had mites and created pouches including Water, LB, Supernatent, E.coli, E.coli RFP. Placed at room temperature and in chris’s drawer.
Took 48 and 24 hour biofilms and performed a bleach treatment on them (0.252%) for 30 minutes. .2015-June-16
Lab book june 16
Names: Kieran
Took oxalic acid readings after 23 hours of incubation
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Title for table Oxalic Acid Concentration (%) OD600 0.0 1.745 0.01 1.771 0.05 1.882 0.1 0.980 0.15 0.125 0.2 0.104 0.3 0.118 0.4 0.174 0.5 0.1456 2.0 0.017 Readings are unrealiable as the cultures were left in the shaking incubator for over 23 hours. Still show the same general trend as past gradients.
2015-June-17
Lab book june 17
Names: Kieran
Make oxalic acid samples again.
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Make 24h biofilms.Title for table Biofilm test (30 minutes contact) (24 hour biofilm) Result Windex Negative Bleach Negative Tilex Biofilm remained 2015-June-19
Lab book june 19
Names: Chris and Tiffany D
Objective:Determine if we have successfully cultured mites in vitro using either paraffin pouches or bee pupae.
Table 2. Experimental design showing each of the treatments applied to the mites.
After 24 hours, all mites were found to be dead. Pupae showed some signs of black bacteria growth or browning on body. Will need to repeat or find another method of culturing mites in vitro.2015-June-22
Lab book june 22
Names: Sydnee, Tiffany D, and Kieran
Biofilm test performed on
Fantastic = Biofilm remained
Windex = Biofilm remained
Bleach = didn’t remain
Made overnight cultures for oxalic acid testing2015-June-23
Lab book june 23
Names: Kieran
13 hours left in shaker
Oxalic Acid Concentration (%) OD600 0.0 1.945 0.01 1.875 0.05 1.763 0.1 0.710 0.15 0.114 0.2 0.146 0.3 0.183 0.4 0.170 0.5 0.092 2.0 0.015 2015-June-25
Lab book june 25
Names: Chris and Ross
Objective:Determine if the concentration of oxalic acid that kills bacteria will kill bees.
Procedure:Bees were obtained from the “Mighty Hive” near the penitentiary at 4:00 and placed into 5ml centrifuge tubes. The bees were transported to the University of Lethbridge and stored at room temperature until 8:00. Prior to experimentation, the tubes were supplemented with a sugar water solution to ensure that the bees would not starve. Following feeding, 2ml of each treatment were added to the 5ml tubes containing the bees and set horizontally along the bench. The abdomen of each bee was immersed in the treatment no matter what orientation the bee took. The bees were observed for 1 hour and subsequently euthanized in 70% EtOH.Treatment Before After none Somewhat lethargic. Drank when prompted. Appeared tired but otherwise unaffected. 0.00% Most lethargic, did not eat when offered. Wet, and slow moving. 0.05% Very vigorous; ran around inside the tube. Fed. Very energetic. Unaffected by treatment. 0.20% Very vigorous; ran around inside the tube. Fed. Slightly less energetic than the 0.05% bee. Table 1. Observations of the bees following 60 minutes of exposure.
Discussion:The bees showed that they could survive well in concentrations of oxalic acid that were above the point where E.coli would die. Further tests must be done to determine the exact contact toxicity. Additionally, the contact toxicity to mites will need to be addressed.
Conclusion:The bees will be able to survive all of the oxalic acid that the E.coli can produce.
2015-June-29
Lab book june 29
Names: Sydnee, Dinula,Tiffany D, and Kieran
Bee: A More Specific Oxalic Acid Gradient
Objective: Determine a more specific concentration of oxalic acid that will prevent cell growth. Already we know that oxalic acid prevents cell growth between 0.05% and 0.2%. Now we want to determine a more specific concentration of oxalic acid that will prevent cell growth.
Oxalic Acid Concentration (%) OD600 0.05 0.07 0.1 0.125 0.13 0.17 0.185 0.2 Cells were left in the shaker for 23 hours accidently. ODs were not taken because they had been left in the shaker too long.
Biofilms: Continued Growth of Biofilms.
Objective: Continue to determine what cleaners will destroy/degrade biofilms. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.
300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.
Results: The biofilms did not grow on any of the plates as there was no red ring. Using fresh RFP cultures might fix this issue because the cultures we had used were prepared earlier in the month.
2015-June-30
Lab book june 30
Names: Kieran, Sydnee, Dinula, Tiffany D, and Marissa
Objective: Due to the oxalic acid gradient from yesterday being left in the shaker too long, the gradient was repeated again.
Oxalic Acid Concentration (%) OD600 0.05 1.810 0.07 1.850 0.1 1.816 0.125 0.130 0.13 0.075 0.15 0.271 0.17 0.231 0.185 0.067 0.2 0.180 Oxalic Acid Concentration (%) pH 0.05 7.90 0.07 7.46 0.1 7.32 0.125 4.56 0.13 3.87 0.15 4.11 0.17 4.01 0.185 3.85 0.2 3.79 Cells were left in the shaker for 15 hours and OD’ed the next morning.
Biofilms: Continued Growth of Biofilms
Objective: The biofilms made yesterday did not work, so fresh RFP cultures were prepared tonight for future biofilm testing. Colonies were picked from a streaked plate of RFP and placed in 5 mL of LB. 5 μL of KAN was added to each tube. Two picked colonies were prepared and left overnight in the 37˚C shaker.
July
2015-July-2
Lab book july 2
Names: Kieran, Sydnee, and Ronja
Prepped bee pouches for in vitro mite/bee feeding.
Performed Bleach and Fantastic testing on RFP biofilms and did one with crystal violet to check for biofilm formation.
Made biofilms for July 3.
2015-July-3
Lab book july 3
Names: Dinula and Chris
Can Bees Ingest Culture?
Objective: Determine if bees can ingest E.coli in liquid culture form with supplementation of sugar. Also, can mites survive on papers that have water and RFP liquid culture?
Procedure: Bees were obtained from the “Mighty Hive” near the penitentiary at 6:50 after a heinous struggle and lack of proper equipment. They were then fed RFP producing E.coli that had been supplemented with both glucose and sucrose sugars. The bees were then observed to determine if they were drinking the solution.
Mites were added to petri dishes following a sugar shake. The dishes had different paper derivatives soaked in treatment; H2O and RFP E. coli. They were then placed in the 27C iGEM incubator. They will be observed in the morning.
The bees were euthanized on EtOH, then surface sterilized in bleach, opened using a razor to expose the gut, and placed in LB to shake at 37C overnight. Problem: 50ul of kanamycin was added instead of 5ul. I suppose that’s what happens when you work a 13 hour day in the sun.
Results:
Treatment Before After none Somewhat lethargic. Drank when prompted. Appeared tired but otherwise unaffected. 0.01% Most lethargic, did not eat when offered. Wet, and slow moving. 0.05% Very vigorous; ran around inside the tube. Fed. Very energetic. Unaffected by treatment. 0.20% Very vigorous; ran around inside the tube. Fed. Slightly less energetic than the 0.05% bee. Table 1. Observations of the bees following 60 minutes of exposure.
Discussion: UPDATE TOMORROW
Conclusion: UPDATE TOMORROW
2015-July-6
Lab book july 6
Names: Sydnee, Andy, Dinula, Ronja, Tiffany D, and Kieran
Objective: To continue growing biofilms and determine if certain cleaners will prevent further growth of the biofilms
Procedure: Same protocol used as June 30. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.
300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.
Bees: Preparing Solutions for Oxalic Acid Gradient using Potassium Permanganate.
Objective: Solutions were prepared to be used for the oxalic acid assay that will see KMnO4 being oxidized by oxalic acid and therefore detect the presence of oxalic acid.
Procedure: To prepare the 1% and 20% Oxalic Acid concentrations, solid oxalic acid was weighed for a solution of 50 mL. 0.5 g of oxalic acid was weighed out and placed in a 50 mL falcon tube. 50 mL of d2H¬2O was added to the falcon tube to dissolve the oxalic acid and create the 1% Oxalic Acid concentration. Two falcon tubes of 1% Oxalic Acid was prepared.
10 g of oxalic acid was then weighed and dissolved in 50 mL of d2H¬2O in a 50 mL falcon tube for the 20% oxalic acid concentration. The 50 mL of 20% oxalic acid was then placed in the 60˚C oven to help dissolve the oxalic acid.
In addition 1 M H2SO4 was created using stock 5 M for the Wieden Lab. Both the 1 M H2SO4 and 1% oxalic acid were stored in the 4˚C fridge in E770.
2015-July-7
Lab book july 7
Names: Sydnee, Tiffany D, and Kieran
Biofilms: Preparing More Biofilms and Reculturing the July 6 Biofilms
Objective: To continue growing biofilms for future testing and to determine if biofilms can be recultured from previous ones that had been degraded based on the July 6 biofilms.
Procedure: New Biofilms
New biofilms were created for July 7 testing. Same protocol used as June 30. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.
300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.
Procedure: Degrading the July 6 Biofilms
The biofilms created yesterday successfully grew. To degrade the biofilms, bleach, Tilex and Truly cleaner solutions were used that had previously been created. The solutions of biofilms were disposed of in the biohazard waste and the microtiter plates placed in water. They were vigoursly shaken to dry and 300 μL of respective cleaning solution added to each well. Since three plates of biofilms were created, each cleaning solution received its on plate. 15 mins after the solutions was added, they were disposed of in the biohazard waste.
Procedure: Reculturing the July 6 Biofilms
To culture the biofilms from yesterday, 300 μL of LB media was added to one well of a microtiter plate. The media was then pipette up and down to mix with any remnants of the biofilms. The media was then pipetted back into the 5 mL tube of LB media. 5 μL of KAN was added to the 5 mL tube of LB media.
Process was repeated for the remaining two plates that used different cleaners.
Note: Before cultures were degraded using the different cleaners, Kieran had taken a sample of the biofilms and prepared a 5 mL tube of LB to culture the biofilm that would be ensured to grow.
Cultures were left to grow 24 hours in 37˚C incubator.
2015-July-8
Lab book july 8
Names: Sydnee, Tiffany D, Dinula, Ronja, Kaatje, and Kieran
Biofilms: Results of July 6 Recultured Biofilms, Degradation of July 7 Biofilms and Preparation of New Biofilms for Further Testing
Objective: Determine if yesterday’s recultured biofilms were successful. In addition continue to degrade biofilms using the household cleaners brought into lab for testing and see if we can successfully reculture the biofilms created yesterday.
Results: Reculturing of the July 6 Biofilms
We were successful in reculturing the biofilms to varying extents based on the overnight cultures of the degraded RFP biofilms. Based on reference of the biofilm that had been cultured before the cleaners were added, the Truly is the most red in colour. Both bleach and Tilex had a more clear appearncec similar to LB.
PHOTO
Procedure: Degrading the July 7 Biofilms
The biofilms created yesterday successfully grew. To degrade the biofilms, Tilex, Truly and Fantastic cleaner solutions were used that had previously been created. The solutions of biofilms were disposed of in the biohazard waste and the microtiter plates placed in water. They were vigoursly shaken to dry and 300 μL of respective cleaning solution added to each well. Since three plates of biofilms were created, each cleaning solution received its on plate. 15 mins after the solutions was added, they were disposed of in the biohazard waste.
Procedure: Reculturing the July 7 Biofilms
To culture the biofilms from yesterday, 300 μL of LB media was added to one well for one of microtiter plate that had been degraded with cleaner. The media was then pipette up and down to mix with any remnants of the biofilms. The media was then pipetted back into the 5 mL tube of LB media. 5 μL of KAN was added to the 5 mL tube of LB media.
Process was repeated for the remaining two plates that used different cleaners.
Note: Before cultures were degraded using the different cleaners, Kieran had taken a sample of the biofilms and prepared a 5 mL tube of LB to culture the biofilm that would be ensured to grow.
Cultures were left to grow 24 hours in 37˚C incubator.
Procedure: New Biofilms
New biofilms were created for July 9 testing. Same protocol used as June 30. Instead of using crystal violet dye, we are using E.coli DH5α cells with RFP (PSB4K5). 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture.
300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.
Bees: Continued Preparation of Solutions for the Oxalic Acid Gradient Using KMnO4
Objective: Continue preparation of solutions being used for the oxalic acid gradients with KMnO4
Procedure
Solutions of 0.01 M of KMnO4 were prepared from stock. 0.158 g of KMnO4 was dissolved in 100 mL of d2H2O. 0.003 M of KMnO4 was then prepared using the 0.01 M solution.
The oxalic acid gradient was also set up with 10 1.5 mL centrifuge tubes with the following oxalic acid concentrations:0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.
A 1 mL volume was created for each concentration using 1% oxalic acid and d2H2O
All solutions and the oxalic acid gradient were set aside for tomorrow on the bench.
2015-July-9
Lab book july 9
Names: Sydnee, Tiffany D, Dinula, Ronja, Jonathan, Aum, and Kieran
Biofilms: Results of July 7 Recultured Biofilms, Degradation of July 8 Biofilms and Preparation of New Biofilms for Further Testing
Objective: Determine if yesterday’s recultured biofilms were successful. In addition continue to degrade biofilms using the household cleaners brought into lab for testing and see if we can successfully reculture the biofilms created yesterday.
Results: Reculturing of the July 7 Biofilms
We were successful in reculturing the biofilms to varying extents based on the overnight cultures of the degraded RFP biofilms. Based on reference of the biofilm that had been cultured before the cleaners were added, the Truly is still the most red in colour. Both Fantastic and Tilex was cloudy with culture but not with the RFP used to originally culture the biofilms.
PHOTO
Procedure: Degrading the July 8 Biofilms
The biofilms created yesterday successfully grew. To degrade the biofilms, Fantastic and bleach cleaner solutions were used that had previously been created. Water was used as a control. 300 μL of respective cleaning solution added to each well. 15 mins after the solutions was added, they were disposed of in the biohazard waste.
Procedure: Reculturing the July 8 Biofilms
To culture the biofilms from yesterday, 300 μL of LB media was added to one well for one of microtiter plate that had been degraded with cleaner. The media was then pipette up and down to mix with any remnants of the biofilms. The media was then pipetted back into the 5 mL tube of LB media. 5 μL of KAN was added to the 5 mL tube of LB media.
Process was repeated for the remaining two plates that used different cleaners.
Cultures were left to grow 24 hours in 37˚C incubator.
Procedure: New Biofilms
New biofilms were created for July 10 testing. 2 μL of KAN was added to 2 mL centrifuge tubes along with 1980 μL LB and 20 μL of culture (E.coli DH5α cells with RFP (PSB4K5)).
300 μL of mix was pipetted into wells of 96 well plate. Biofilms left to grow 24 hours in 37˚C incubator.
Bees: Oxalic Acid Gradient Using KMnO4 –July 9
Object: To determine for the presence and concentration of oxalic acid present in solution using the oxidation of KMnO4
Procedure
1.Standard oxalic acid solution (1 mg/mL) was prepared with distilled water
2.100 mL of 0.003 M KMnO4 was prepared from appropriate dilution of 0.01 M KMnO4 in distilled water
3.500 mL of 2 N H2SO4 was prepared in distilled water
4.Assay mixtures contained different concentrations of oxalic acid ranging from 0.1 to 1.0 mg of oxalic acid (we had 1 mL samples of different percentage concentrations of oxalic acid), 5 mL 2 N H2SO4, and 2 mL of 0.003 M KMnO4
5.This mixture was incubated for 10 mins at room temperature (27˚C)
6.After 10 mins absorbance was recorded at 528 nm on the spec in E720
7.Reagent blank was prepared with distilled water
8.The calibration curve obtained is linear in concentration range of 0.1 mg to 1 mg/mL of oxalic acid
9.The regression equation is ∆A = 0.966C -0.027 with correlation coefficient 0.983 where C is the concentration of oxalic acid in mg/mL and ∆A is Ab-As
Data
Title for table Oxalic Acid Concentration (%) OD528 0.1% 1.487 0.2 -0.005 0.3 -0.005 0.4 -0.003 0.5 0.6 -0.006 0.7 -0.007 0.8 -0.004 0.9 -0.005 1.0 -0.008 2015-July-10
Biofilms: Degradation of July 9 Biofilms and Reculturing of July 9 Biofilms
Names: Sydnee, Tiffany D, Dinula, Ronja, and Kieran
Objective: Continue to degrade biofilms using the household cleaners brought into lab for testing and to see if we can successfully reculture the biofilms.
Procedure: Degrading the July 8 Biofilms
The biofilms created yesterday successfully grew. To degrade the biofilms, Fantastic and Lysol solutions were used that had previously been created. Water was used as a control. 300 μL of respective cleaning solution added to each well. 30 mins after the solutions was added, they were disposed of in the biohazard waste.
Procedure: Reculturing the July 9 Biofilms
To culture the biofilms from yesterday, 300 μL of LB media was added to one well for one of microtiter plate that had been degraded with cleaner. The media was then pipette up and down to mix with any remnants of the biofilms. The media was then pipetted back into the 5 mL tube of LB media. 5 μL of KAN was added to the 5 mL tube of LB media.
Process was repeated for the remaining two plates that used different cleaners.
Cultures were left to grow 24 hours in 37˚C incubator.
2015-July-10
Ligation
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2015-July-10
Ligation
We did this stuff...
2015-July-10
Ligation
We did this stuff...
2015-July-10
Ligation
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2015-July-10
Ligation
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