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

Revision as of 05:13, 14 October 2018

Notebook

Week 1 (May 1-4): Completed lab safety training (WHMIS and Biosafety Level 1 Certification was completed by all wet lab members), 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: Facebook video.

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, practiced transformation, liquid culture preparation, plate streaking, and miniprep techniques.

Week 5 (May 28-June 1):

• Used electroporation to transform 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 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, and control mix)
• 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 → 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
  • Transformed nanoluc containing pNL1.1 plasmid 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
• Performed Gibson Assembly to insert an Anderson Promoter into pNL1.1 for the luciferase assays
• 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
  • One of three pET16b digestions was as expected 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.
  • Luciferase assay confirmed production of NanoLuc was occuring
• Plated liquid cultures on kanamycin and kanamycin + cortisol plates
  • No growth

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.

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
• Ordered Nanoluc containing sequence from Pnl1.1 with biobrick prefix,suffix and illegal restriction sites removed

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
• Completed the InterLab Measurement Study this week
• Gibson Assembly of Nanoluc Coding Device, into DH5a

Week 13 (July 23-27):

• Calibrated Luminometer with Nanoluc Coding device. Used serial dilutions of NanoLuc containing solution, found decreasing readings with luminometer corresponding to the relative decrease

July 25th: Second SynBio Club Meeting

July 26th: Science Quest Mentorship

Week 14 (July 30-August 3):

• grew up more pET16b and linearized it (digested) for Gibson assembly
• Made liquid cultures of 4HT Kan, and Ampicillin stocks and plates.
• Transformation of 3-2ER intein into BL21. Selected colonies, performed mini-preps with isopropanol to remove DNA-ase. Gel confirmed succesful transformation
• Assembled a FRET biosensor using acGFP and mCherry, then expressed it in BL21 DE3, tested it in a fluorometer and found some promising results. For example, we had fluoresce of 8 billion AU in some wells, compared to 700 million in the LB controls

Week 15 (August 7-10):

• GFP Autosplicing Intein construct did not work. Confirmed extensively with LB controls.
• FRET based biosensor might be working, on fluorometer there was huge increases of MCherry emission compared to LB controls, when excited at 475nm (acGFP excitation)
• Composite BioBrick consisting of:a Anderson Promoter - Ribosome Binding Site - NanoLuc® - Terminator works! consistent Nanoluc production. Characterized time course kientics
• First attempt at error-prone PCR unsuccessful, positive control did work. Suspect primers are too long. Ran a gradient still no result. Tm suspected to be too high. Will redesign

Week 16 (August 13-17):

• FRET biosensor works! Tested on a fluoremeter and found 150× increase over a water control, then he went, then to the Biomedical confocal imaging Laboratory at Queen’s and found that the antagonist reduced the FRET interaction. (A.K.A., this proves that the biosensor works!)
• ER intein tested in BL21 on 4-HT plates, still no results, even with good controls. Was concerned the ethanol which the 4-HT is dissolved in might be killing the bacteria but its not the case
• Digested FRET, confirmed insert size with gel

Week 17 (July 30-August 3):

• Took another visit to the biomedical imaging laboratory this week to take quantitative images of the FRET biosensor. He took photos of the FRET biosensor under cortisol, mifepristone, and ethanol (control) conditions (while keeping the magnification, excitation, and filtration constant). Now will begin analyzing the relative intensity of GFP and mCherry fluorescence in each image to assess the amount of FRET occurring.
• Tested the ER intein being expressed in BL21, by inducing expression with IPTG and incubating for 4 hours at 37°C, then added 4-HT, incubated for 1 more hour, then plated 200 μL on plates containing 10 μM 4-HT and kanamycin, and another on kanamycin alone. No growth
• oGrew up 10 mL of BL21 expressing the same intein construct, then induced expression, and 4 hours later separated the 10 mL into 5 mL with 4-HT, and 5 mL without. Then lysed the cells and collected the cell lysate, the soluble fraction, and the cell debris portion. These were then mixed with 50 μL of Coomassie blue (dye for Western blotting) and frozen it down
• We also attempted Error Prone PCR on the intein this week, however it wasn't successful

Week 18 (August 6-10):

• Ran an SDS for the protein fractions from last week•It showed that the intein is being made (large band at 77 kDa)! But, it’s insoluble, which Dr. Petkovich suspected and explains why there was no splicing producing kanamycin resistance

Week 19 (August 20- 24):

• No lab work
• Attended Queen's in the park event to recruit students

Week 20 (August 27- 31):

• No work done this week, wiki infrastructure in place.

Week 21 (September 3rd - 7th):

• Attempted to Clone FRET and ER-Intein into pSB1C3 from gel extracted EcoRI and PstI. T4 ligation at 30 mins room temp, then 30 mins in fridge. Chemical transformation, but no prepared SOC so used LB. No colonies grew
• Ran a gel on the Ligation products, looked positive, there was band forming around 5kb, indicative of succesful ligaton, but transformation didnt work
• Presented at Engineering Design Fair

Week 22 (September 10th - 14th):

• No Updated

Week 23 (September 17th - 21st):

• Cloning FRET and Intein re attempted with NEB 5-alpha compotent cells, no success!!
• We want to clone the intein cause although it didnt work, it improves characterization of a previous part. I.e The intein being tested in bacteria instead of yeast.

Week 24 (September 24th - 28st):

• Growing up liquid culture of Nanoluc coding device/Pnl1.1 with illegal cut sites removed.
• Liquid culture, mini prep, ran gel. Confirmed insert is present.

Week 25 (Oct 1st - 5th):

• No luck with cloning. Even tried using very new T4 ligase. Gel extracted pSB1C3 from mRFP, and used linearized pSB1C3 provided by igem then PCR clean up.

Week 26 (Oct 8th - 12th):

• NanoLuc coding device added to registry, then sent to igem on DNA submission Deadline
• Added FRET and ER- Intein too because they are good chracterizations of parts, even if we couldnt clone them into pSB1C3