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Harvard iGEM 2019 Lab Notebook



*A note on attribution of work: having a team of 3, very often two or three of us would work together on the same task, and so it would be often not be very helpful to write attributions for each task; we assume practically equally distributed work, and hope this is sufficient for the competition’s purposes.


JULY 2

  • Ran PCRs of GFP-Actin, mNeonGreen, BFP-NLS inserts, and pDUO backbones

  • Performed ZymoClean of PCR products.

  • NanoDrop concentrations: GFP-Actin mNeonGreen BFP-NLS Insert 78.4 ng/µL 360.7 ng/µL 280.6 ng/µL Backbone (pDUO) 188.9 ng/µL 175.6 ng/µL 98.7 ng/µL ○ Gibson assembly of each insert/backbone pair: 10 µL 2x Mastermix + 300 ng insert, 300 ng backbone, balance sterile dH2O

  • Incubation at 50 °C for 60 min

  • Gel of PCR products showed none of expected bands

  • 1% agarose, 90 V, 60 min

JULY 3

  • Ran PCRs of mNeonGreen (mNG) with myristoylation tag (Myr), palmitoylation tag (Palm), and endoplasmic reticulum (ER) localization tag (1st transmembrane domain of Aquaporin 1), plus corresponding backbones (pDUO)

  • All inserts at 61 °C with Phusion High-Fidelity PCR MasterMix (HF Buffer), all backbones at 64 °C with Q5 High-Fidelity 2x MasterMix

  • Performed ZymoClean of PCR products

JULY 4

  • Repeated PCRs of mNG with Myr, Palm, and ER tags, as well as Hygromycin resistance gene ○ Performed ZymoClean of PCR products

  • NanoDrop concentrations Myr-tagged mNG Palm-tagged mNG ER-tagged mNG HygR Insert 159.2 ng/µL 110.7 ng/µL 97.5 ng/µL 183.5 ng/µL Backbone (pDUO) 372.9 ng/µL 344.8 ng/µL 396.7 ng/µL n/a

  • JULY 8

  • Ran PCRs of mNeonGreen-Myr, mNeonGreen-Palm (both 64 °C and 66 °C attempted), GFP-actin from Gibson assembly and transformation July 2nd

  • Performed NanoDrop on above insert PCRs, BFP-NLS insert and backbone, and GFP-Actin insert and backbone from July ____ Myr-tagged mNG Palm-tagged mNG GFP-Actin colony Insert low temp 95.0 ng/µL 213.6 ng/µL Insert 341.5 ng/µL Insert high temp 142.2 ng/µL 244.5 ng/µL BFP-NLS GFP Insert 165.9 ng/µL 217.4 ng/µL Backbone (pDUO) 492.7 ng/µL 597.7 ng/µL

  • Ran overnight PCR of leftover mix for Myr-tagged mNG and Palm-tagged mNG inserts

JULY 9

  • Ran overnight PCR of Myr- and Palm- tagged mNG in gel and performed ZymoClean

  • Only ER insert has been successfully PCRd, plan to Gibson assemble together ER insert, gBlock with Aquaporin1 transmembrane domain, and ER backbone

  • Inoculated successful BFP-NLS colony in 2 mL LB-Blasticidin broth

JULY 10

  • Performed MiniPrep of overnight BFP-NLS colony culture, roughly 16 hours post-inoculation

  • Performed colony PCRs of ER-tagged mNeonGreen for backbone, and GFP-Actin backbone

  • Amplifying pDUO containing mNeonGreen or GFP-Actin around intended BFP-NLS insertion site, for subsequent Gibson assembly with amplified BFP-NLS inserts

  • NanoDrop concentrations of colony backbone PCRs: ER-tagged mNG backbone (w/ BFP-NLS) GFP-Actin backbone (w/ BFP-NLS) 133.5 ng/µL 65.0 ng/µL

  • Ran Gibson assembly of BFP-NLS backbones

  • Inoculated BFP-NLS-containing pDUO cells in LB-Blasticidin for overnight culture

JULY 12

  • MiniPrep of overnight BFP-NLS culture

  • NanoDrop concentration of plasmid isolate: 261.6 ng/µL

  • Ran insert PCR checking for BFP-NLS from MiniPrep product

  • Used SYBR green gel, noted that GelRed gives better looking bands

  • Band for ER-tagged mNG backbone, but none for

JULY 13

  • Ran backbone PCR for GFP-Actin insertion into pDUO

  • Ran gel of PCR product (lanes 6 and 7)

  • Gibson assembly of GFP-actin and pDUO containing BFP-NLS

  • 75 ng backbone (175.3 ng/µL) + 225 ng insert (217.4 ng/µL), 10 µL 2x Gibson mastermix, 8.6 µL sterile dH2O

  • Neg. control: backbone (pDUO + BFP-NLS), mastermix, water

  • Transformation with Gibson product

  • 10 µL Gibson product added to 16.6 uL competent cells (DH5-alpha E. coli)

  • 40 minutes recovery time in SOC broth before plating

  • Negative control and GFP-Actin transformed cells plated on same Blasticidin plate, one group per side

JULY 14

  • Moved plates transformed 7/13 to 4 °C

JULY 15

  • PCRs:

  • 3 PCRs of colonies picked from plates transformed 7/13

  • GFP-Actin insert, ER-tagged mNG insert, GFP-Actin backbone (pDUO + BFP), BFP insert PCR from backbone (pDUO + BFP)

JULY 16

  • PCR of BFP-pDUO colonies (numbers 4, 5, and 6) for insert

  • Colonies 5 and 6 have bands 700-1000 bp (expected)

  • Began overnight culture of colonies 5 and 6 in 2 mL Blasticidin LB broth at 4:30 pm

JULY 17

  • Performed MiniPrep of overnight cultures of BFP-NLS colonies 5 and 6 (10:30 am)

  • NanoDrop concentrations, MiniPrep plasmid products:

  • Colony 5: 251.8 ng/µL

  • Colony 6: 282.6 ng/µL

  • Ran re-try PCRs of GFP-Actin insert, ER-tagged mNG insert

  • PCRs of BFP-NLS miniprep, of both BFP-NLS “insert” and pDUO backbone, for colonies 5 and 6 miniprep products

  • Gel: stcrong band for GFP-Actin insert, none for ER-tagged mNG, or BFP-NLS miniprep insert (either colony)

JULY 18

  • Performed PCR of Er-tagged mNG insert

  • Phusion MasterMix

  • Tried gradient of temperatures as troubleshooting mechanism

  • 4 reactions at temperatures from 56 °C to 66 °C

  • None of the mNG template worked, GFP backbone insert (a.k.a. BFP-NLS) PCR (Miniprepped colony 6) had a correct band

  • Troubleshooting PCR of mNG template (“insert”)

  • Gradient PCR, Q5 MasterMix, 59 °C to 67 °C (3 temperatures, 2 templates)

  • ZymoClean of GFP-Actin backbone (old BFP colony 5), GFP-Actin insert, mNG-ER backbone (old BFP colony 6)

  • all PCR products, in other words

  • NanoDrop concentrations:

  • GFP-Actin backbone: 378.2 ng/µL

  • GFP-Actin insert: 293.9 ng/µL

  • mNG-ER backbone: 166.2 ng/µL

  • Gibson assembly: GFP-Actin backbone (pDUO) and GFP-Actin insert

  • into MCS2

  • 80 ng vector, 100 ng GFP-actin insert

  • 10 uL mastermix, 9.5 uL dH2O, 0.3 µL GFP-A insert, 0.21 µL GFP-A BB

  • Prepared LB-agar + Blasticidin

  • 1 packet Blasticidin added to 200 mL milliPore water (in fume hood!) and LB-agar block within 500 mL bottle

  • Repeated microwaving until dissolved - lost some volume due to boiling over - may have increased Blasticidin concentration due to undissolved Blasticidin still present

  • Aliquoted 1 mL per well in 9 12-well plates, made one dish (circa 25 mL)

  • Transformed DH5 alpha competent cells with Gibson product (GFP-Actin BB + insert)

  • 30 minutes on ice, 42 °C for 30 sec, SOC broth addition, 37°C shaking recovery (1 hour) ○ Ran gel of the 6 mNG-ER template (insert) PCRs

JULY 19

  • At 10:30 am, colonies (from GFP-Actin/BFP-NLS/pDUO transformation) look possibly present but clear-ish, small (not very distinguishable from water droplets or air bubbles)

  • 11:15 am: slightly bigger, more irregular and opaque

  • Picked 3 colonies, swirled (using sterile wooden picks) in 100 µL sterile dH2O

  • Colony PCRs: 3x(GFP-Actin insert, BFP-NLS insert)

  • 25 µL Q5 MM, 2.5 µL fwd primer, 2.5 µL reverse primer, 1 µL cell suspension in water, 19 µL dH2O

  • 3 min denaturation/cell destruction step (98 °C), annealing 62 °C

  • Ran on gel (order: ladder, GFP-Actin for colonies: 1,2,3, BFP-NLS for colonies: 1,2,3)

  • All GFP inserts successful and similar band intensity; BFP-NLS inserts present on colonies 1 and 3 (though 3 quite faint)

  • Started 2 liquid cultures derived from colony 1 water swizzle (GFP-A, BFP-NLS) in 2 mL LB + Blasticidin (4:45 pm) START OF MECHANOSENSOR PROJECT WORK

JULY 22

  • Yuki provided new mNeonGreen template! Streaked out

  • Mechanosensor project primer plate arrived

  • Resuspended primers to 100 µM

  • 100x µL as nmol present, per manufacturer spreadsheet

  • HEK293T still at 30-40% confluency by early afternoon (not ready for transfection using GFP-Actin/BFP-NLs pDUO

  • Backbone PCRs: pDUO w/ beta-arrestin/TEV protease primers (Ta = 64 °C), pSecTag2 w/ Piezo1 backbone primers (Ta = 71 °C), pDUO w/ Calmodulin 1 primers (Ta = 63 °C)

  • 50 µL rxns (25 µL Q5 MM, 2.5 µL each primer, 1 µL template (pDUO/pSecTag2, 19 µL dH2O)

  • Unfortunately...none of these backbone PCRs worked!

  • ZymoClean yields: 30 ng/µL, 5 ng/µL, 9 ng/µL

JULY 23

  • PCR of backbones for Piezo1, Calmodulin 1, and β-arrestin-TEV protease

  • Temperatures:

  • Piezo1: 67.6 °C, 69.4 °C, 71.9 °C

  • CaM1: 60 °C, 65.7 °C, 67 °C

  • β-arrestin-TEV: 60.7 °C, 64.4 °C, 67 °C

  • Extension time: 3 min

  • Gel order:

  • lane 5:

  • ladder (GeneRuler 1 kb Plus per usual) / Lanes 6-15: Piezo1 T1, T2, T3, etc for CaM1 & β-arrestin-TEV

  • No Piezo1 backbone bands, bands for T2, T3 for CaM1, all temperatures β-arr backbone

  • ZymoClean yields (most intense bands cleaned):

  • β-arr T3: 282.8 ng/µL

  • CaM1 T3: 515 ng/µL

  • Gibson Assembly (gBlocks designed with overhangs already included so no need to perform insert PCRs)

  • 80 ng backbone (pDUO)

  • 85.18 ng β-arr gBlock template (2035 bp)

  • 21.64 ng CaM1 gBlock template (5734 bp)

  • 20 µL rxns

  • Negative control for each: no insert, only backbone

  • Transformation (chemically competent, same procedure)

  • Seeded HEK293T cells into 96-well plate, 12-well plate, and T-75 flask in preparation for transfection with assembled pDUO + GFP-Actin + BFP-NLS

  • 100,000 cells/mL seeding density, 100 uL seeding volume for 96-well plate, 2 mL seeding volume of 12-well plate

  • Wells D4-9 in 96-well plate

  • 4 wells in 12-well plate

  • Overnight cultures (2 mL LB-amp): Yuki’s streaked mNeonGreen cells

  • 20 µL Amp stock + 1980 µL LB broth

JULY 24

  • MiniPrep of mNeonGreen cells grown overnight

  • MiniPrep product yield: 364.9 ng/µL

  • Massive run of PCRs, missing documentation

  • Lipofectamine 3000 standard protocol

  • Diluted DNA mix:

  • GFP-Actin/BFP-NLS pDUO: 1.96 µL DNA + 65 µL OptiMEM + 2.6 µL P3000

  • Control: 1.96 µL water + 65 µL OptiMEM + 2.6 µL P3000

  • 65 µL DNA mastermix + 65 µL Lipofectamine mastermix (3.9 µL Lipofectamine reagent + 130 µL OptiMEM)

  • 10 µL per 96-well well, 500 µL per 12-well well

JULY 25

  • Miniprep of CaM1-pDUO transformed cells cultured overnight (to be backbone for Calcineurin)

  • Miniprep product yield: 218.4 ng/µL

  • ZymoClean: Piezo1 backbone PCR product (pSecTag2 + overhangs)

  • Ran PCR of Calcineurin backbone (with Calmodulin insert), from Miniprep product ■ Annealing temp: 65 +/- 3 °C

  • Accidentally added CaM1 backbone to Gibson β-arr tube - have to redo Gibson rxn for β-arr template/backbone (+ negative control)

  • Same volumes/rxns as on July 23rd

  • Transformations:

  • Piezo1 (3 gBlocks, 4 fragments including backbone) + pSecTag2 ( + neg control)

  • Β-arr + pDUO ( + neg control)

  • Plated transformed bacteria in 24-well Hygromycin plates

  • Streaked out on Amp plates reporter plasmids (pre-modification) ordered from AddGene, with 5xUAS and TRE-tight promoters (4:20 pm)

  • Changed media (DMEM + 10% FBS) to phenol red-free media for 96- and 24- well Lipofectamine-transfected HEKs (transient transfection)

  • On 96-well plate, all wells had weird polyp-shaped, yellowish mass of cells - Tim had never seen something like this, possible contamination but looked like HEK morphology on cell level

  • Mentor Tim Chang imaged transfected HEKs around midnight Transfected (overlaid blue/green filter) Untransfected (overlaid blue/green filter)

  • Absence of blue signal confirms initial BFP-NLS sequencing results (no priming)

JULY 26

  • Left lid to 24-well Hygromycin plate slightly open, agar dried up and nothing grew - have to repeat transformation

  • Colonies on reporter plasmid plates (Amp)

  • Transformation volumes:

  • 5 µL Gibson rxn (frozen -> thawed) + 17.5 µL competent cells

  • Re-ran Calcineurin backbone PCRs T1-3 on gel (because dye seemed to have diffused out of gel)

  • CaN BB PCRs T1 and T2 had bands, proceeded to ZymoClean these

  • T2: 666.2 ng/µL

  • Started Gibson rxn with backbone T2 and Calcineurin gBlock (overhangs already on)

  • Began liquid culture in LB broth-amp of colonies from plated 5xUAS and TRE-tight reporters (2:15 pm)

  • Plated re-transformed cells (β-arr, Piezo1 + negative controls) on Blasticidin plate around 2 pm, as well as Piezo1 on Amp plates (as should only have Amp resistance)

  • Preparation of chemically competent cells

  • Started overnight culture of previously competent E. coli at 4:15 pm

  • 50 µL competent cells thawed from -70 °C + 5 mL LB broth

JULY 27

  • Miniprep of 5xUAS and TRE cells grown overnight - 10:00 am (a little late, after 16 hours maximum recommended culture time)

  • Miniprep product concentrations:

  • 5xUAS: 338.7 ng/µL

  • TRE-tight: 162.2 ng/µL - low

  • Began competent cell subculture (0.5 mL overnight culture + 50 mL LB) at 2:15 pm

  • Passaged HEK293T cells

  • 9.0e5 cells seeded in T75

  • OD600 (optical density) of competent cell subculture at 2:35 pm: 0.35

  • Ran PCRs: BFP backbone (from 5xUAS miniprep July 27th), GFP backbone (from pTRE-tight miniprep July 27th)

  • OD600 at 3:20 pm: 0.68

  • NEB protocol recommended putting cells on ice at OD 0.5-0.7 but other protocols say .3-.4, .4-.6 etc - confusing

  • Put cells on ice, centrifuged 10 min 4000 rpm (4 degrees C) in 2 x 50 mL centrifuge tubes

  • 15 mL 30 mM CaCl2 : 49.94 mg in 15 mL dH2O

  • Distributed 1.5 mL resuspension to 6 eppendorf tubes and spun 30 seconds (microcentrifuge)

  • Resuspended each pellet in 0.5 mL ice-cold 30 mM CaCl2 in cold room, without vortexing - flicked each tube to mix

  • Aliquoted 50 µL into many 0.5 mL eppendorf tubes, flash-froze and stored -70 degrees C

  • Started liquid culture of Cam1+CaN (pDUO colony)

  • NOTE: FORGOT GLYCEROL!

JULY 28

  • Miniprep of liquid culture CaM1/Can pDUO

  • Nanodrop: 495.7 ng/µL

  • Changed media for HEK293T cells plated July 27th

  • Thawed one of competent cell aliquots, transformed with GFP/BFP (pDUO) plasmid (BFP should not show in insert PCR but GFP should and/or should grow on blasticidin plate)

  • ZymoClean of PCR products:

  • BFP backbone: 330.6 ng/µL

  • mNeonGreen backbone: 537.5 ng/µL

  • β-arrestin backbones (1 and 2): 130.6 ng/µL and 133.8 ng/µL

  • Calcineurin A backbone: 274.2 ng/µL

  • Piezo1 backbone: 279.5 ng/µL

JULY 29

  • Both BFP/GFP (pDUO) wells on competent cell test have lots of colonies - competent cells seem to be prepared properly/working

  • PCRs:

  • BFP insert, mNeonGreen insert, colony GFP insert (x2 colonies)

  • BFP insert has correct band -> ZymoClean (Nanodrop 282.4 ng/µL)

  • mNeonGreen insert has no band - redo PCR w temperature gradient and new template dilution (still from Miniprep of Yuki’s cells)

  • Gibson assembly rxns:

  • BFP insert + BFP backbone (6647 bp) - 15 min

  • 3:1 insert:vector - 23.76 ng insert:75 ng vector

  • Β-arrestin insert + β-arrestin backbone (pDUO) - 15 min

  • 3:1 insert:vector - 85.18 ng insert:75 ng vector

  • Piezo1 insert + Piezo1 backbone (pSecTag2 - 4991 bp) - 60 min (4 fragments)

  • 2:1 insert:vector - 91.74 ng insert:75 ng vector

  • + 3 negative controls (no inserts)

  • Transformations: 16.6 µL competent cells + 10 µL Gibson rxn mix

JULY 30

  • Re-transforming all from yesterday as well as a positive control

  • Used last aliquot of Tim’s competent cells and began overnight culture of 5 µL cells in 2 mL LB at 4:15 pm

  • Neglected to add glycerol to competent cell preparation on July 27th

  • Hypothesis: some survived at low temp for a day or so and that is how we got positive plasmid presence for our test of the cells with BFP/GFP pDUO - which explains why none of the cells transformed yesterday grew on any of the plates

  • PCRs: colony PCR (Piezo1 x 3 (7.26.19), Piezo1 negative control) - from Amp plates

  • All reactions at 64 °C

  • β-arrestin backbone (38 repeats as opposed to 35 - trying to improve yield)

  • mNeonGreen w/ Phusion MasterMix - final attempt

  • BFP backbone (of “A” template)

  • Transformed:

  • β-arrestin + negative control, Piezo1 + negative control, BFP + negative control

  • Experimental groups: 10 µL cells + 8 µL DNA

  • Negative control groups: 5 µL cells + 4 µL DNA

  • Positive controls: GFP/BFP (pDUO) - Blasticidin, empty pSecTag - Amp

  • No mNeonGreen PCR worked, even using Phusion MM

  • Patrick thinks lab may have mNeonGreen with different sequence than that we used to design primers

JULY 31

  • Chemically competent cell preparation protocol thanks to iGEM Duesseldorf 2018 (Author: Anna Behle): https://www.protocols.io/view/preparation-of-chemically-competent-cells-n8mdhu 6

  • Started subcultures of 2 overnight cultures of competent cells at 10:30 am

  • Redundancy because otherwise out of chemically competent cells!

  • Colony numbers from transformation July 30th Piezo1 0 Piezo1 C- 0/1 5xUAS-BFP 60+ 5xUAS-BFP C- 4 β-arrestin 3 β-arrestin C- 0 pSecTag empty (C+) 100+ BFP/GFP pDUO (C+) 100+

  • OD600 : 11:23 am 12:10 pm 12:30 pm Subculture 1 0.09 0.23 0.34 Subculture 2 0.09 0.24 0.35

  • At OD600 of roughly 0.35, transferred cells to 2 sterile 50 mL Falcon tubes and incubated on ice for 20 min (always kept cells cold from this step onward)

  • Prepared CaCl2 solution (100 mM): 45 mL dH2O + 499.4 mg calcium chloride

  • 85 mM calcium chloride + 15% glycerol

  • Centrifuged cells for 5 min 4500 rpm and 4 degrees C and discarded supernatant

  • Carefully resuspended each pellent in 20 mL of ice-cold 100 mM calcium chloride, incubated on ice for 60 min

  • Centrifuged for 5 min at 4500 rpm and 4 degrees C and discarded supernatant

  • Very carefully suspended each pellet in 2 mL ice-cold 85 mM calcium chloride with 15% glycerol, then aliquoted out into .5 mL tubes in cold room

  • 5xUAS BFP successful colony cultured overnight (2 mL LB-Amp) beginning 5:45 pm

  • Overnight PCRs: Piezo1 colony PCR, Piezo1 backbone (pSecTag2), B-arrestin gBlock, B-arrestin (pDUO)

AUGUST 2

  • Ran gel of PCR w troubleshooting conditions

  • Β-arrestin insert:

  • Temperatures (degrees Celsius): 64, 66.5, 68

  • For each temperature, one reaction with DMSO, one without

  • Piezo1 colonies (insert):

  • Colonies 1-8

AUGUST 3-7

  • More retries of Piezo1 cloning cycle: PCR, Gibson assembly, transformation

AUGUST 8

  • Colony PCRs for Piezo1

  • Ta = 71 C, 5:30 extension time

AUGUST 12

  • Tim has no HEK293T cells growing already, can’t transfect CaM1/CaN (pDUO) plasmids

  • Backbone PCRs: for BDKBR2, GPR68 (pDUO)

  • Ta = 67 °C

  • x2 each (using fresh and old dilutions of “pDUO New”

  • All had bands, but between 7 kb and 10 kb - strange

  • Gibson assembly rxns:

  • BDKBR2

  • 75 ng vector, 87.11 ng insert

  • GPR68

  • 75 ng vector, 82.76 ng insert

  • Negative controls for both (vector alone)

SEPTEMBER 13

  • PCRs: Number Gibson rxn role Source Primer plate wells Annealing temp (°C) 3 “GFP backbone” pTRE B10, B11 62 4 “GFP insert” GFP/BFP pDUO B8, B9 63 7 “Piezo1 backbone” pSecTag2 Old plate 67 8 “Piezo1 insert” gBlock B11, A4 9 “GPR68 backbone” Addgene plasmid (with B-arr) C2, C3 62 10 “GPR68 insert” GAL4-VP16 gBlock B12, C1 58 11 “B-arr backbone” Addgene plasmid (with B-arr) C6, C7 62 12 “B-arr insert” pDUO C4, C5 58 (Blasticidin resistance)

OCTOBER 17

  • Lent one-lane channel from Scott - may be able to use to set up experiment, but hopefully can find multilane device

  • Transfection groups: n = 3

  • Bradykinin and Yoda1 (chemical agonists for BDKBR2 and Piezo1 respectively) - ogerin (GPR68 agonist) could not ship from last week in time

OCTOBER 18

  • Began overnight cultures of:

  • BDKBR2-tTa, beta-arrestin/TEV protease, GPR68 “vanilla” (tTa rather than Gal4-VP16), mPiezo1 (mouse Piezo1) “vanilla”, 5xUAS-luciferase “vanilla” (unmodified)

  • Cultures from at least 2 colonies each for redundancy

OCTOBER 19

  • Miniprepped (6 am, +18 hr) all overnight cultures from previous night

  • Yields: Colony BDKBR2 Beta-arrestin mPiezo1 GPR68 “vanilla” 1 67.7 ng/µL 456.9 ng/µL 289.3 ng/µL 461.7 ng/µL 2 88.0 ng/µL 286.5 ng/µL 453.4 ng/µL 68.5 ng/µL 3 335.8 ng/µL

  • Other plasmids (previously Miniprepped)

  • Calmodulin 1/Calcineurin (pDUO) - sequenced

  • NFAT-mScarlet

  • “3/4 C2” (pTRE)

  • “9/10 C1” (GPR68-GAL4-VP16)

  • BFP-UAS

  • BFP positive control (from Tim)

  • Groups (n=3): 1 BDKBR2-tTa, beta-arrestin-TEV protease, pTRE (GFP) 2 pTRE (GFP) 3 mPiezo1, NFAT/mScarlet, Cam1/CaN 4 mPiezo1, NFAt/mScarlet 5 NFAT/mScarlet 6 GPR68-Gal4-VP16, beta-arrestin-TEV protease, UAS (BFP) 7 GPR68-tTa, beta-arrestin-TEV protease, pTRE (GFP) 8 UAS (BFP) 9 BFP positive control ○ Had seeded and been growing for 4 days HEK293T cells in rows D, E, F, columns 1-12 of 96 well plate ○ Each column corresponds 1-9 to group, rows D and E to be treated with agonist ○ Transfection: 0.2 µg total DNA for each well (Lipofectamine 3000 protocol) - mass split between number of plasmids per group

  • Added 10 µL/well of final P3000/Lipofectamine/DNA/OptiMEM mixtures to each well for appropriate group

  • Incubated roughly 20 hours

OCTOBER 20

  • BDKBR2 stimulation with Bradykinin:

  • Dissolved 10 mg bradykinin acetate salt to 25 mg/mL in 0.1 M acetic acid, diluted in DMEM+10% FBS for stimulation “dose” of 1µL, at 50 nM in wells

  • mPiezo1 stimulation with Yoda1

  • Dissolved 5 mg Yoda1 powder in 1.4 mL DMSO to obtain 3.55 mg/mL stock

  • Treated cells at 50 µM with mixture of Yoda1 DMSO solution and DMEM+10% FBS

  • Rows D and E received agonist, F did not - swapped media before adding

  • Save cells in row E for passaging into multilane microfluidic channel

  • Treated GPR68 with bradykinin (agonist for BDKBR2), because did not have other use for GPR68 cells without agonist ogerin, and given hypothetical possibility of partial/weak activation by BDKBR2

  • After 60 minutes incubation with agonists, removed media and added 150 µL PBS (also to reduce autofluorescence of medium [e.g. with phenol red]) during fluorescence imaging

  • Imaging information:

  • Widefield fluorescence

  • Filters: DAPI (for BFP), Alexa 488 (for EGFP), Alexa 568 (for mScarlet)

  • Acquired images of each well, saved one of 9 subsections of image per well ○ With apparently no mPiezo1 activation leading to signal, decided to incubate 1 day further with agonist present

  • Removed PBS in wells D and E, added 150 µL DMEM+10% FBS + agonist as before

  • However, changed Bradykinin concentration from 50 nM to 500 nM (10x)

  • Received multilane PDMS device (10 lanes) from Scott!

  • Roughly 100 µm x 75 µm main flow channel, nonsterile, more hydrophobic than ideal due to being fabricated 2018

  • Passaged cells from row E (transiently transfected), seeded on order of 50 µL resuspended pellet

  • Pellet invisible due to exceedingly small number of cells - difficult to ensure retention, which may have led to tiny number of cells visibly seeded in microfluidic channels

  • Seeded using long thin gel loading tips ○ Incubation begun 11:30 pm

OCTOBER 21

  • Removed DMEM+10% FBS from rows D, E, replaced with PBS, imaged as per October 20

  • Unfortunately, microfluidic channels did not have more than a few cells visible at confocal 4x or 10x objective - according to Carlos (Paulsson Lab) need confluency to not push cells out of chamber with flow - decided experiment was unrealistic, very time-constrained, and anyway infeasible with present cell growth

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