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.

April

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May

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June

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July

• 2015-July-10
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August

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September

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• June

• 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

  .

  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.
  
  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 testing

• 2015-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.

• 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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