Team:Northern BC/Notebook
Northern BC iGEM 2019 Lab Notebook
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May 2019
May 24: Michael
Prepared 25 LB + Carb plates.
May 28: Michael
Received in kind gifts from Dr. Stephen Rader (UNBC):
Placed yeast strains streaked on a YPD plate in the 30 ˚C incubator (1:30PM)
Started overnight cultures of bacterial gifts to make glycerol freezers stocks
•5 mL LB + 5 µL 1000X Carb (final [carb] = 50 µg/mL) + 1 colony
•flame sterilized culture tube every time something was added
•shook on incubator at 300 rpm, 37 ˚C overnight
May 29: Michael
Prepared liquid LB media.
Made glycerol stocks of bacterial strains
•500 µL overnight culture + 250 µL 50% glycerol
•snap frozen in liquid nitrogen and stored at -80 ˚C
Prepped plasmid from the remaining liquid cultures using the NEB Monarch®
Plasmid Miniprep Kit (NEB Cat# T1010S)
Nanodrop plasmid preps with the Nanodrop ND-1000
May 30: Michael
Tested fresh LB + Carb plates
•Streaked DH5 alpha (-) and DH5 alpha + pRS423 (+) and incubated the plate at 37 ˚C overnight (1PM)
Assessed purity of plasmids prepped yesterday by agarose gel electrophoresis
•Prepared a 1% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide
•Loaded 100 ng of each plasmid purified yesterday
May 31: Michael
Test plates look great! No growth on the (-) and plenty of growth on the (+).
June 2019
June 12: Sahej, Lisa, Jasneek
Made fresh 1 mL aliquots of 1000X Carbenicillin.
Prepared 1 L of 10X TBE Gel Running Buffer.
Made 500 mL liquid LB media.
June 21: Sahej
Made 300 mL 1M Lithium Acetate.
Made 100 mL 50% Glycerol.
Made 100 mL 10X TE buffer.
28 June 2019 Sonia, Mohamed, Bushra
Made 1 L liquid YDP media.
July 2019
July 02: Jasneek, Lisa, Michael
Made 5 M Sodium chloride (5 M NaCl) stocks.
First round of site-directed mutagenesis to remove RFC-10 restriction enzyme sites from the yeast shuttle vectors, pRS423 (PstI) and pRS425 (EcoRI).
Reactions were set up as follows:
Program:
Stage 1: 98 °C for two minutes
Stage 2: 98 °C 10 seconds (denature)
54 °C 45 seconds (primer annealing)
72 °C 10 minutes (extension)
Repeat stage two for 18 cycles
Stage 3: 72 °C 20 minutes
Stage 4: 4 °C indefinitely
PCR products were stored at -20 ˚C overnight.
July 02: Sonia
Inoculated 10 mL YPD media with a single colony of BY4741 and incubated on a
shaker overnight at 30 ˚C, 250 rpm. This is in preparation to analyze the
effect of methanol on the growth rate of our yeast strain to determine what the maximum allowable methanol carry-over can be in our downstream biosensor testing since the samples we intend to test are solubilized in methanol.
July 03: Lisa, Jasneek, Michael
Treated site-directed mutagenesis PCR products with DpnI (NEB Cat# R0176S).
Added 1 µL DpnI (20 units) directly to the PCR products and incubated in a water bath at 37 ˚C for four hours. Start: 10 AM, End: 2 PM.
Ethanol precipitate DpnI-treated reactions to clean-up and concentrate DNA.
•added 50 uL ddH2O, 5 uL 5 M NaCl,1 uL 20 mg/mL glycogen, and 1 mL cold 100% ethanol.
•let stand in the -20 ˚C freezer for 30 minutes.
•spun in a microcentrifuge at 13,000 rpm at 4 °C for at least 30 minutes.
•poured off the ethanol and gently added 1 mL cold 70% ethanol to wash the pellet (do not resuspend the pellet).
•spun again as above but for only five minutes.
•removed all ethanol and air-dried pellet on the bench for approximately 15 minutes.
Transformed the whole pellet into 50 µL RbCl2 competent DH5 alpha.
•gently resuspended DNA with competent cells.
•place tube on ice for 30 minutes.
•heat shock at 42 ˚C for 90 seconds.
•without shaking the tubes, transferred to ice and left for two minutes.
•added 100 µL LB media and placed on a shaker for 30 minutes at 37 ˚C.
•plated entire reaction on LB + Carb plate.
•incubated plates at 37 ˚C overnight.
July 03: Sonia
BY4741 starter culture was not saturated enough to start the methanol growth
assays. Will need to re-start. Inoculated 100 mL liquid YPD with 5 mL of
starter culture and incubated at 30 ˚C, 250 rpm overnight.
Made 25 YPD plates and 12 LB + Chloramphenicol plates.
July 03: Sahej
Ran a diagnostic restriction enzyme digest using pRS425 as a template to test the efficiency of our EcoRI-HF (NEB Cat# R3101S) and SpeI-HF (NEB Cat# R3133S) enzymes.
Plasmid was mini-prepped by Michael on 29 May 2019 (pRS425-2).
Used cutting reaction recipe for linearized backbones from iGEM help page.
Also prepared an uncut control by mixing 1 µL template DNA with 24 µL nuclease-free water.
•Incubated at 38 ˚C for 3 hours
•Heat inactivated at 65 ˚C for 20 minutes
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•added 5 µL of 6X Gel Loading Dye, Purple (NEB – supplied with restriction enzymes)
•ran 100 bp DNA ladder, 120V
Result: Both enzymes appear to be cutting well. The gel could have run longer as the ladder is not even separated. Pattern is a little unusual.
July 04: Jasneek, Michael
Selected 5 colonies from each site-directed mutagenesis transformation plate and also two colonies from a DH5 alpha + pUC19 plate to screen by RE-digest.
Grew each colony in 5 mL LB + 5 µL 1000X Carb, shaken overnight at 37 ˚C, 250 rpm.
July 05: Michael, Jasneek
Prepped plasmid from the overnight liquid cultures using the NEB Monarch®
Plasmid Miniprep Kit (NEB Cat# T1010S) for all but one pf the DH5 alpha + pUC19 cultures, which was used to test an old QIAprep Spin Miniprep Kit (Qiagen Cat# 27104).
Nanodrop plasmid preps with the Nanodrop ND-1000.
Results: The Qiagen miniprep kit contents have gone bad. Discard this kit.
pRS425-2 and pRS425-4 are no good. Discard the tubes.
All other minipreps look good. Proceed to screen for successful elimination of the PstI site from pRS423 and the EcoRI site from pRS425 by restriction enzyme digest.
Incubated in a water bath at 37˚C.
Digest start: 1:32 PM; end: 2:30 PM.
Added 10 uL of loading dye to each reaction and prepared an uncut control for both pRS423-1 and pRS425-1 by adding 1 µL of uncut plasmid to 24 µL of ddH2O followed by the addition of 5 µL of loading dye.
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•ran 1 kb DNA ladder
•gel start 3:08 PM, end 3:53 PM. 120V
Expected outcome:
If the PstI site has been successfully removed from pRS423, we should see one band running at ~5800 bp; if unsuccessful we will see two bands, one at 950 bp and the other at 4850 bp.
If the EcoRI site has been successfully removed from pRS425, we should see one band running at ~6850 bp; if unsuccessful we will see two bands, one at 2050 bp and the other at 4800 bp.
Results: We have successful mutagenesis (pending sequencing for final confirmation).Going forward we will name pRS423-4 ‘pGEM12’ and pRS425-1 ‘pGEM17’. For each there is a clear single band located at approximately the position we would expect to see linearized plasmid for each.
Begin second round of site-directed mutagenesis to remove XbaI restriction site from pGEM12 and pGEM17. Reactions were set up as follows:
Program:
Stage 1: 98 °C for two minutes
Stage 2: 98 °C 10 seconds
54 °C 45 seconds
72 °C 10 minutes
Repeat stage two for 18 cycles
Stage 3: 72 °C 20 minutes
Stage 4: 4 °C indefinitely
PCR products were stored at -20 ˚C overnight.
July 06: Jasneek
Treated site-directed mutagenesis PCR products with DpnI (NEB Cat# R0176S).
Added 1 µL DpnI (20 units) directly to the PCR products and incubated in a water bath at 37 ˚C for four hours. Start: 10 AM, End: 2 PM.
Ethanol precipitate DpnI-treated reactions to clean-up and concentrate DNA.
•added 50 uL ddH2O, 5 uL 5 M NaCl,1 uL 20 mg/mL glycogen, and 1 mL cold 100% ethanol
•let stand in the -20 ˚C freezer for 30 minutes
•spun in a microcentrifuge at 13,000 rpm at 4 °C for at least 30 minutes
•poured off the ethanol and gently added 1 mL cold 70% ethanol to wash the pellet (do not resuspend the pellet)
•spun again as above but for only five minutes
•removed all ethanol and air-dried pellet on the bench for approximately 15 minutes
Transformed the whole pellet into 50 µL RbCl2 competent DH5 alpha.
•gently resuspended DNA with competent cells
•place tube on ice for 30 minutes
•heat shock at 42 ˚C for 90 seconds
•without shaking the tubes, transferred to ice and left for two minutes
•added 100 µL LB media and placed on a shaker for 30 minutes at 37 ˚C
•plated entire reaction on LB + Carb plate
•incubated plates at 37 ˚C overnight (19 hours)
July 07: Jasneek
Selected 5 colonies from each site-directed mutagenesis transformation plate to screen by RE-digest. Grew each colony in 5 mL LB + 5 µL 1000X Carb, shaken overnight at 37 ˚C, 250 rpm.
July 08: Jasneek and Michael
Prepped plasmid from the overnight liquid cultures using the NEB Monarch® Plasmid Miniprep Kit (NEB Cat# T1010S).
Nanodrop plasmid preps with the Nanodrop ND-1000.
Results: pGEM12-3 and pGEM17-5 are no good. Discard the tubes. All other minipreps look good. Proceed to screen for successful elimination of the XbaI (NEB Cat# R0145S) site by restriction enzyme digest.
Incubated in a water bath at 37˚C.
Digest start: 1:32 PM; end: 2:30 PM.
Added 10 uL of loading dye to each reaction and prepared an uncut control for both pRS423-1 and pRS425-1 by adding 1 µL of uncut plasmid to 24 µL of ddH2O followed by the addition of 5 µL of loading dye.
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•ran 1 kb DNA ladder
•120V
Expected outcome:
If the XbaI site has been successfully removed from pGEM12, we should see one band running at ~5800 bp; if unsuccessful we will see two bands, one at 3200 bp and the other at 2600 bp.
If the XbaI site has been successfully removed from pGEM17, we should see one band running at ~6850 bp; if unsuccessful we will see two bands, one at 3200 bp and the other at 3650 bp.
Results: Unsuccessful.
Restart the XbaI site-directed mutagenesis as before.
July 10: Michael
No colonies on either the pGEM12 or pGEM17 XbaI site-directed mutagenesis plates. Set this PCR up again as before.
July 11: Lisa
Treated site-directed mutagenesis PCR products with 1 uL DpnI (NEB Cat# R0176S) for 5 hours at 37 ˚C and stored at -20 ˚C overnight.
July 12: Michael
Ethanol precipitated and transformed DpnI digest products as before. Plated on LB + Carb and placed in the incubator at 37 ˚C overnight.
July 13: Jasneek
Only two colonies on the pGEM13 plate and eleven colonies on the pGEM18 plate. Set up overnight cultures for both pGEM13 candidates and five pGEM18 candidates, each in 4 mL LB + 4µL 1000X Carb. Left to shake for 16 hours.
July 14: Lisa, Jasneek
Prepped plasmid from the overnight liquid cultures using the Wizard® Plus SV Minipreps DNA Purification Systems Kit (Promega Cat# A1470). Eluted in 100 µL nuclease-free water.
Nanodrop plasmid preps with the Nanodrop ND-1000.
Results: pGEM12-3 and pGEM17-5 are no good. Discard the tubes. All other minipreps look good. Proceed to screen for successful
elimination of the XbaI (NEB Cat# R0145S) site by restriction enzyme digest.
Incubated in a water bath at 37˚C.
Digest start: 1:55 PM; end: 2:55 PM.
Added 10 uL of loading dye to each reaction and prepared an uncut control for both pRS423-1 and pRS425-1 by adding 1 µL of uncut plasmid to 24 µL of ddH2O followed by the addition of 5 µL of loading dye.
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•ran 1 kb DNA ladder
•120V, 40 min
Expected outcome:
If the XbaI site has been successfully removed from pGEM12, we should see one band running at ~5800 bp; if unsuccessful we will see two bands, one at 3200 bp and the other at 2600 bp.
If the XbaI site has been successfully removed from pGEM17, we should see one band running at ~6850 bp; if unsuccessful we will see two bands, one at 3200 bp and the other at 3650 bp.
Results: We have successful mutagenesis of pGEM12, resulting in pGEM13 (pending sequencing results). Unfortunately, none of the pGEM18 candidates were successful.
Set-up six more overnight cultures to screen additional pGEM18 candidates.
July 15: Jasneek
Prepped plasmid from the overnight liquid cultures using the Wizard® Plus SV Minipreps DNA Purification Systems Kit (Promega Cat# A1470). Eluted in 100 µL nuclease-free water.
Nanodrop plasmid preps with the Nanodrop ND-1000.
Results: Minipreps look good. Proceed to screen for successful elimination of the XbaI (NEB Cat# R0145S) site by restriction enzyme digest.
Incubated in a water bath at 37˚C.
Digest start: 12:50 PM; end: 1:50 PM.
Added 10 uL of loading dye to each reaction and prepared an uncut control for both pRS423-1 and pRS425-1 by adding 1 µL of uncut plasmid to 24 µL of ddH2O followed by the addition of 5 µL of loading dye.
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•ran 1 kb DNA ladder
•120V, 1 hr
Expected outcome:
If the XbaI site has been successfully removed from pGEM17, we should see one band running at ~6850 bp; if unsuccessful we will see two bands, one at 3200 bp and the other at 3650 bp.
Results: Colony 1, 2 and 4 were not successful, but colonies 3, 5, and 6 appear to be successful (pending sequencing).
Start an overnight culture to prep more pGEM13.
July 16: Jasneek, Michael
Transform pGEM18-5 into DH5alpha to make more plasmid.
Prepped pGEM13 from the overnight liquid cultures using the Wizard® Plus SV Minipreps DNA Purification Systems Kit (Promega Cat# A1470). Eluted in 100 µL nuclease-free water.
Nanodrop plasmid preps with the Nanodrop ND-1000.
July 17: Jasneek, Michael
PCR gene inserts.
Program:
Stage 1: 98 °C for two minutes
Stage 2: 98 °C 10 seconds
55 °C 30 seconds
72 °C 3 minutes
Repeat stage two for 29 cycles
Stage 3: 72 °C 20 minutes
Stage 4: 4 °C indefinitely
Start an overnight culture to prep fresh pGEM18-5.
July 18: Jasneek, Michael
Prepped pGEM18-5 from the overnight liquid cultures using the Wizard® Plus SV Minipreps DNA Purification Systems Kit (Promega Cat# A1470). Eluted in 100 µL nuclease-free water.
Nanodrop plasmid preps with the Nanodrop ND-1000.
Set up the following digests:
Incubated digests at 37 ˚C. Start: 9:56AM; End: 11:32 AM. Heat inactivate at 65˚C for 20 minutes.
Prepared a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide.
•loaded 2 µL of each reaction + 3 µL ddH2O + 1 µL gel loading dye
•also loaded 2 µL of uncut pGEM13 + 3 µL ddH2O + 1 µL gel loading dye and 2 µL of each PCR product + 3 µL ddH2O +
1 µL gel loading dye
•ran 1 kb DNA ladder
•120V, 1 hr
Results: The analytical gel shows exactly what was expected: The three PCR products (gGEM1, gGEM2, and gGEM3) have a dominant band at the expected size; the pGEM13 single digests show all three enzymes are working well, and the double digests, relative to the uncut plasmids, appear to have worked as well.
Load the remaining double digests into a 0.7% agarose gel in 0.5X TBE with 0.5 µg/mL ethidium bromide to gel purify the backbone.
Gel purify with the Wizard® SV Gel and PCR Clean-up Systems Kit (Promega Cat# A9281). Also cleaned-up gGEM1, gGEM2, and gGEM3 PCRs.
July 22: Michael
Ligate gGEM2 in to pGEM18 in a 3:1 molar ratio of insert to vector. Not sure if the top or bottom pGEM18 band is the one we want, so ligate insert into both.
Ligate gGEM1 in to pGEM13 in a 3:1 molar ratio of insert to vector.
Left reaction on ice. Start: 1:30PM, End: 7:10PM
Transform ligation reactions into 50 µL competent DH5 alpha.
July 23: Jasneek
There are two colonies on the backbone alone plate, 2 colonies on the pGEM18 bottom band plate, and 45 colonies on the pGEM18 top band plate.
Made 4 x 100 mL LB and 25 LB + Carb plates.
July 24: Jasneek, Michael
Screened colonies for successful ligation by double restriction enzyme following plasmid miniprep. All candidates were empty vector.
Restart ligations.
July 26: Michael, Lisa, Jasneek
Screened candidates for successful ligation. Again, all vectors were empty.
Start preparing more vector and insert as before to try the ligations again.
July 28: Michael, Lisa, Jasneek
Unsuccessful ligations again. Need to figure out the problem.
Problem identified: restriction enzyme sites are too close together. While the single digests work well, the double digest is likely a mixed population of single digests as there is not enough room left for the second enzyme to dock once the first has cut. Will need to re-design strategy and order new supplies.
July 29th Michael
Ran a gel (Agarose 0.7%, EtBr) to verify that the vectors to be ligated into are in fact cut.
July 30 Jasneek
RE digest of CYC1 & OPRMI cleaned up
Inserts & pGEM 18 colony 5
Ran a gel to ensure pGEM18 colony 5 cut
Results: RE digest was successful due to observing 2 bands in the approximate location where they should be.
Broth Cultures of OPRMI + pGEM13 & CYC1 + pGEM18 (Round 2)
Made 5 (5 mL + 1 uL carb) broth cultures for OPRMI + pGEM13 ligation reaction
Made 1 (5 mL LB + 1 uL carb) broth culture for CYC1 + pGEM18 ligation reaction
July 31 Lisa and Jasneek
Bacterial transformation of OPRMI + pGEM13 & CYC1 + pGEM18
Miniprep of pGEM18 + CYC1 and pGEM13 + OPRM1
Used the Promega miniprep kit and followed the kit protocol
1 miniprep of pGEM18 + CYC1 and 5 minipreps of pGEM13 and OPRMI
RE Digest of pGEM13 + OPRM1 Ligation and pGEM18 + CYC1
August 2019
August 1 Michael
Site directed mutagenesis PCR
After analyzing the sequence of the mutated pGEM18 the modified EcoRI site seemed to have a repeated section that was not there before, as if part of the primer inserted itself. Need to re-do this mutation starting back from pGEM3.
PCR set-up/program is identical to before.
August 2 Lisa and Michael
DpnI Digest
Added 1uL DpnI to the PCR reaction for pGEM3 EcoRI site making pGEM17
Incubated at 37℃
Ethanol Precipitation / Transformation
PCR cleaned-up the site directed mutagenesis reaction making pGEM17
Followed the same protocol as before(“Precipitate DNA to Concentration and Clean-up”).
Transformed the plasmid.
August 8 Sahej and Lisa
Made new patch of S. cerevisiae BY4741 on YPD
Incubated at 30℃
Gel purification of pGEM17 cut plasmids and pGEM13 and OPRM1 + CYC1 inserts
Bacterial transformation of pGEM17
Followed bacterial transformation protocol as done previously
0.5uL plasmids used
Bacterial transformation for part BBa_k1073024 to test chloro+LB plates
Followed bacterial transformation protocol as previously done
1uL part BBa_k1073024
August 9 Sonia
Minipreps of pGEM13-1 and pUC19
Followed Promega Wizard Plus SV Miniprep DNA Purification System Protocol
August 10 Sonia
RE-Digest for inserting MCS into pGEM13-1
Prepping backbone pGEM13-1 #1, concentration = 240.0ng/uL (miniprepped Aug 9, 2019)
Aiming to digest 5ug of vector as per Liz’s recommendation
August 11 Lisa and Jasneek
Bacterial transformation for both OPRM1 and CYC1 ligations
Followed bacterial transformation protocol
Transformed in 50uL DH5alpha cells, and incubated at 37℃
Results: both plates had growth on them.
August 13 Jasneek
Miniprep of CYC1 & OPRM1 overnight ligations and OPRM1 2 hour ligation
Followed Promega Wizard Plus SV Miniprep DNA Purification System protocol.
All species were RE-digest to confirm the ligation was successful except the CYC1 + pGEM17 overnight 1 due to low plasmid concentration.
August 14 Lisa and Jasneek
PCR Amplification of CYC1 Insert and OPRM1 Cassette
Broth Cultures for pGEM13 and pGEM17
Ethanol Precipitation of PCR Products (OPRM1 and CYC1)
Followed ethanol precipitation protocol
August 19 Sonia
Transformation (lacZ) + Cultures
August 21 Jasneek and Sonia
PCR Clean Up & EtOH Precipitate of CYC1 & OPRMI Inserts
Followed precipitate DNA to concentrate & clean up protocol
Gel Purification and Extraction
Gel 2 = Gel purification (followed the Promega Wizard SV GEL and PCR Cleanup
pGEM13 and MCS Ligation
September 2019
September 9 Jasneek and Sonia
Ligation of CYC1 Insert into pGEM17-1
Ligation of MCS in pGEM13
September 21 Sonia
Ligation of pGEM13 and MCS
Made LB and YPD broth and inoculated cultures
Inoculated 6, 5mL LB+carb broths with ligation colonies from day before
Broths inoculated for 18 hours at 300rpm and 37 ℃
Miniprepped pGEM14 broths and performed as per Promega Wizard Plus SV Miniprep DNA purification systems but final elution was eluted into two separate 50uL elutions
September 23 Sahej
PCR of KanMX
Diluted previous KanMX PCR 1:10 such that it’s ~100 ng/uL for future PCR
PCR Cleanup of KanMX using ethanol precipitation
September 24 Sonia
RE-digest of pGEM14 colonies:
Digested pGEM14 - 1.1: (30uL total + 6uL 6X GLD)
Digested pGEM14 - 1.2: (30uL total + 6uL 6X GLD)
Digested pGEM14 - 1.4: (30uL total + 6uL 6X GLD)
Digested pGEM14 - 2.2: (30uL total + 6uL 6X GLD)
September 25 Sahej
Followed Promega Wizard Plus SV Miniprep DNA purification System protocol to miniprep pGEM13. Eluted into 2x 50uL nuclease-free water elutions.
RE-digest of pGEM13 and KanMX PCR cleanup (for 4 hours):
Digest KanMX: (20uL total)
Digested pGEM13: (30uL total)
September 25 Sonia
Analytical gel
Loaded (from left to right): 1kB NEB ladder, Cut pGEM14 - 1.1, cut pGEM14 - 1.2, cut pGEM14 - 1.4, cut pGEM14 - 2.1, cut pGEM14 - 2.2
September 26 Lisa
Made LB/carb broth cultures of pGEM17 (3mL LB + 3uL carb), and grown overnight as described previously.
September 27 Sonia
Miniprep performed using Promega protocol as described previously.
RE-Digest of pGEM17-1 + CYC1/aeBlue Insert
Also ran a control pGEm17-1 with same reaction recipe.
September 30 Jasneek
Screening of CYC1 Ligation into pGEM17-1
Mixed 3uL of each of the 10 candidate plasmids with 12uL mastermix and incubated for 1 hour at 37 °C
Ran digested candidate plasmids on a gel, as follows, left to right: 1kB Ladder (NEB), Candidate plasmids 1-10, 1kB Ladder (NEB)
Ran a DpnI digest for 4.5 hours, then ethanol precipitated it as described previously. After, transformed into 50uL DH5alpha cells.
Completed PCR to mutate the XbaI site in pGEM17-1, using the same thermocycler settings as described previously.
