Difference between revisions of "Team:Queens Canada/Notebook"

Revision as of 10:16, 22 August 2018

Notebook

Week 1 (May 1-4): Completed lab safety training (WHMIS and Biosafety Level 1 Certification was completed by all wet lab members), finished and compiled all relevant protocols on Benchling, signed up for Interlab Measurement Study, began ordering necessary reagents.

May 6th: Attended Canadian Undergraduate Technology Conference (CUTC) hosted by the University of Waterloo and presented at our own booth at the Maker’s Fair

Week 2 (May 7-11): Ordered all necessary reagents and worked on developing our DNA sequence & attended QICSI Bootcamp courtesy of the Dunin-Deshpande Queen’s Innovation Centre

Here’s a short clip from the weekend pitch competition our team participated in, held at the Donald Gordon Conference Centre.

May 12th: Participated in Science Rendezvous held at the Rogers K-Rock Centre, where we had our own booth featuring fruit DNA extraction, a microscope, and a VR headset with a virtual look into what is found inside cells.

Week 3 (May 14-18): Our team was selected as one of ten teams to be awarded an OT-2 Automatic Pipetting Robot courtesy of Opentrons Labworks

See more information here.

Sequences have been ordered as gblocks. Will be ordering kanamycin resistance construct for the estrogen receptor as a positive control, one with the glucocorticoid receptor, and three different versions of the same construct with linkers of varying length and flexibility between the LBD and the intein.

Week 4 (May 22-25):Prepared media and ampicillin plates, obtained our plasmid, learned cloning, liquid culture preparation, plate streaking, miniprep and nanodrop techniques.

Week 5 (May 28-June 1):

• Used electroporation to amplify pET16b within our host E. coli strain, DH5-alpha
• 10 µL and 100 µL of the bacteria was plated on ampicillin plates and stored at 37°C overnight
• Prepared liquid cultures using four random colonies from the ampicillin plates
• Performed a plasmid MiniPrep to isolate plasmid DNA. Concentrations were determined using UV spectrophotometry
• Performed a diagnostic restriction digest using HindIII and AvaI and ran on agarose using gel electrophoresis
• Following confirmation of successful transformation, bacterial glycerol stocks were prepared (stored at -80°C)

Week 6 (June 4-8):

• Made chemocompetent cells of DH5-alpha and BL21
• Resuspended gblock-NanoLuc-gblock DNA
• Linearized our plasmid with BamHI and XhoI
• Performed a PCR gel extraction to ensure the plasmid insert and other cell debris were removed
• Used the NanoDrop to determine concentrations of both the DNA and plasmid
• Performed Gibson Assembly on the linearized plasmid and the gblock NanoLuc, as well as performed a negative control of Gibson Assembly® Master Mix by substituting DNA with water
• Used electroporation in 3 DH5-alpha samples (Gibson Assembly® Master Mix, diluted Gibson Assembly® Master Mix according to actual Gibson instructions and control mix without DNA)
• 10 µL and 100 µL of each sample were plated on ampicillin plates and colony growth was observed where expected

Week 7 (June 11-15):

• Prepared 4-hydroxytamoxifen + kanamycin plates and cortisol + kanamycin plates
• Used a restriction digest to test the NanoLuc Gibson Assembly product
  • Results were inconclusive → tubes used had limited oxygen and prevented bacterial growth from occurring
• Colonies were re-grown, MiniPrep was done, restriction digest was performed again
  • Results were inconclusive → gel was run at too high of a voltage
• Chloramphenicol plates were made for Interlab Measurement Study while waiting for DNA sequences to arrive
• Learned how to perform luciferase assays

Week 8 (June 18-22):

• Started the week with InterLab Measurement study:
  • Made standard curves for fluorescein, microsphere particles, and LUDOX CL-X using a plate reader
  • Attempted the cell measurement protocol:
    • Started by making 40 chloramphenicol plates. Then heat-shock-transformed DH5-alpha with the plasmid, then plated
  • The next day, all plates were coated with a lawn of growth, including the negative control
    • Cells were competent, but the chloramphenicol that we used was expired (first opened in 2011); the antibiotic would have broken down and was no longer selective. Will have to attempt again
• Transformed a plasmid called pNL1.1 into DH5-alpha using chemical transformation
• Grew a liquid culture Thursday night of DH5-alpha housing pNL1.1, and another liquid culture of DH5a with pET16b
• Performed plasmid mini-prep (extracted the plasmid DNA) and did a double digest of each KpnI and EcoRI for pNL1.1, XhoI and HindIII for pET16b
• Attempted Gibson Assembly to insert an Anderson Promoter from Genscript into pNL1.1 for the luciferase assays (check out QGEM 2017 for more info on Anderson Constitutive Promoters)
• Performed another Gibson assembly to insert 2 overlapping fragments into pET16b:
  • N-terminal of KanR
  • N-intein-LBD (GR)-C-intein-KanR
  • Did electroporation to transform E. coli and plated
  • The Gibson Assembly for pNL1.1 didn’t work (currently still promoterless)
    • Nothing grew (including on the positive control) → therefore, could be a plasmid, transformation, or Gibson assembly problem

Week 9 (June 25-29):

• Gibson was redone with a HiFi Gibson Assembly on pNL 1.1 and the Anderson promoter, as well as pET16b and intein with three different linkers (GPGGSG, GPGGSGS, GGGGSGGGGS), and a positive control
• Did chemical transformation using New England Biolabs supplies and plated on ampicillin plates
  • Colonies were recovered on every plate (including the positive control)
  • Now have colonies for pNL1.1 +Anderson promoter and for the three different sequences with KanR+intein+linkers+GR domain
• Made liquid cultures
• Performed a diagnostic digest on three linkers using BsaI
  • Results were not as expected, only digested pET16b with linker 1 came out perfectly as expected on the gel
• Performed a luciferase assay on pNL1.1:
  • Pelleted 1-2 mL of liquid culture bacteria (pNL1.1). Repeated (four different liquid cultures).
  • Added 1 mL of lysis buffer (contains: lysozyme, β-mercaptoethanol, phosphate buffered saline (PBS)).
  • Let sit for 30 min.
  • Sonicated the mix for 11 seconds, then put on ice. Repeated twice.
  • Pelleted cellular debris by centrifuging, and collected the supernatant (contains the protein).
  • Ran a serial dilution in a white 96-well plate (1×, 0.5×, 0.25×, 0.125×).
  • Repeated for four rows of different liquid cultures. Added one extra for a negative control.
  • Results were not as expected, β-mercaptoethanol (reducing agent) in lysis buffer was believed to have interfered with substrate of protein
• Plated liquid cultures on kanamycin and kanamycin + cortisol plates
  • Expected to see cells growing in presence of cortisol but not in the absence → opposite results were obtained, cells were growing only on kanamycin plates but not in the presence of cortisol

June 29th: First SynBio Meeting

Week 10 (July 3-6):

• Tuesday: Prepared plates with ampicillin and cortisol to test if the cortisol was the reason the bacteria wasn’t growing
• Made new SOC media, which will be used in future transformations
• Plated linkers and pNL1.1 from the bacterial stocks for further cortisol and splicing testing
• Wednesday: Made liquid cultures of the plates
• Thursday: Plated all of the liquid cultures on kanamycin plates with varying concentrations of cortisol (10nM, 1 µM, 10µM, 100µM)
• Also plated on ampicillin and cortisol plates with a concentration of 10 µM (and on kanamycin as a control)
• Friday: Results → There were no colonies on any of the plates with cortisol and kanamycin. As well, pNL1.1 grew on the ampicillin and cortisol plates, confirming that cortisol is not killing the bacteria. There were colonies on the kanamycin plates – pNL1.1, linker 1, linker 2, linker 3. This result is unexpected, as pNL1.1 should not grow on the kanamycin plates as it doesn’t have resistance

Week 11 (July 9-13):

• Grew up liquid cultures of our plates (from bacterial stock) to check for potential contamination
• Performed a plasmid MiniPrep and diagnostic digest
  • No contamination of pNL1.1 or pET16b linkers was seen
• Sent DNA on Wednesday for sequencing (the GR, intein, linkers ×3)
• Did Gibson on GFP-intein-GR content, but the pET16b was not as clear in our gel as we would have liked
• Made more cortisol, 4-hydroxytamoxifen, and kanamycin plates for further tests when DNA arrives

Week 12 (July 9-13):

• Performed Gibson Assembly this week for our part, and also now have assembled the glucocorticoid receptor (GR) with NanoLuc and without intein, estrogen receptor, and retinoic acid receptor with a kanamycin resistance (saw growth on these plates as well, and made stocks)
• Ordered primers, dNTPs, and manganese chloride for use in error-prone PCR
• Made a GFP-Intein-GR construct to see if it was truly auto-splicing
• Tested this GFP-Intein-GR auto-splicing (“off switch”) construct, which we believe to only splice in the absence but not presence of cortisol. We tested it this Friday by making a fluorescence measurement in the morning then adding a serial dilution of cortisol and testing again after a six hour incubation. Believed that autosplicing of GFP is occurring, however the results from the test were inconclusive and a longer incubation time will need to be used in further tests
• Completed the InterLab Measurement Study this week

Week 13 (July 23-27):

July 25th: Second SynBio Club Meeting

July 26th: Science Quest Mentorship

Week 14 (July 30-August 3):

Week 15 (August 7-10):

Week 16 (August 13-17):