iGEM 2019 — Team Northern_BC

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# Team:Northern BC/Notebook

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TEAM

[Team Members](https://2019.igem.org/Team%3ANorthern_BC/Team)

[Attributions](https://2019.igem.org/Team%3ANorthern_BC/Attributions)

[Sponsors](https://2019.igem.org/Team%3ANorthern_BC/Sponsors)

PROJECT

[Description](https://2019.igem.org/Team%3ANorthern_BC/Description)

[Design](https://2019.igem.org/Team%3ANorthern_BC/Design)

[Collaborations](https://2019.igem.org/Team%3ANorthern_BC/Collaborations)

WET LAB

[Notebook](https://2019.igem.org/Team%3ANorthern_BC/Notebook)

[Experiments](https://2019.igem.org/Team%3ANorthern_BC/Experiments)

[Recipes](https://2019.igem.org/Team%3ANorthern_BC/Recipes)

[Parts](https://2019.igem.org/Team%3ANorthern_BC/Parts)

[Results](https://2019.igem.org/Team%3ANorthern_BC/Results)

[Safety](https://2019.igem.org/Team%3ANorthern_BC/Safety)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3ANorthern_BC/Human_Practices)

[Public Engagement](https://2019.igem.org/Team%3ANorthern_BC/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Northern_BC)

# Northern BC iGEM 2019 Lab Notebook

###### Jump to: [May](#May) [June](#June) [July](#July) [August](#August) [September](#September)

## May 2019

**May 24:**   *Michael*
[Prepared 25 LB + Carb plates](https://2019.igem.org/Team%3ANorthern_BC/Recipes#LB).

**May 28:**   *Michael*
Received in kind gifts from Dr. Stephen Rader (UNBC):

![](https://static.igem.org/mediawiki/2019/7/7a/T--Northern_BC--Table_1.png)

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.](https://2019.igem.org/Team%3ANorthern_BC/Recipes#LB)
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

![](https://static.igem.org/mediawiki/2019/5/5a/T--Northern_BC--Table_2.png)

**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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Carb).
Prepared 1 L of [10X TBE Gel Running Buffer](https://2019.igem.org/Team%3ANorthern_BC/Recipes#TBE).
Made 500 mL [liquid LB media](https://2019.igem.org/Team%3ANorthern_BC/Recipes#LB).

**June 21:**   *Sahej*
Made 300 mL [1M Lithium Acetate](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Lithium).
Made 100 mL [50% Glycerol](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Glycerol).
Made 100 mL [10X TE buffer](https://2019.igem.org/Team%3ANorthern_BC/Recipes#TE).

**28 June 2019**   *Sonia, Mohamed, Bushra*
Made 1 L liquid [YDP media](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Yeast).

## July 2019

**July 02:**   *Jasneek, Lisa, Michael*
Made 5 M [Sodium chloride](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Sodium) (5 M NaCl) stocks.
First round of [site-directed mutagenesis](https://2019.igem.org/Team%3ANorthern_BC/Experiments) to remove RFC-10 restriction enzyme sites from the yeast shuttle vectors, pRS423 (PstI) and pRS425 (EcoRI).

Reactions were set up as follows:

![](https://static.igem.org/mediawiki/2019/1/13/T--Northern_BC--Table_3.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Experiments#transformation) 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](http://parts.igem.org/Help%3AProtocols/Linearized_Plasmid_Backbones).
Also prepared an uncut control by mixing 1 µL template DNA with 24 µL nuclease-free water.

![](https://static.igem.org/mediawiki/2019/2/27/T--Northern_BC--Table_4.png)

              •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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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.

![](https://static.igem.org/mediawiki/2019/e/e3/T--Northern_BC--Table_5.png)

**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.

![](https://static.igem.org/mediawiki/2019/e/e9/T--Northern_BC--Table_6.png)

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.

![](https://static.igem.org/mediawiki/2019/0/0c/T--Northern_BC--Table_7.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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.

![](https://static.igem.org/mediawiki/2019/2/29/T--Northern_BC--Table_8.png)

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:

![](https://static.igem.org/mediawiki/2019/8/80/T--Northern_BC--Table_9.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Experiments#transformation) 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.

![](https://static.igem.org/mediawiki/2019/5/58/T--Northern_BC--Table_10.png)

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.

![](https://static.igem.org/mediawiki/2019/c/c3/T--Northern_BC--Table_11.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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.

![](https://static.igem.org/mediawiki/2019/c/c4/T--Northern_BC--Table_12.png)

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.

![](https://static.igem.org/mediawiki/2019/b/ba/T--Northern_BC--Table_13.png)

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.

![](https://static.igem.org/mediawiki/2019/2/23/T--Northern_BC--Table_14.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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.

![](https://static.igem.org/mediawiki/2019/3/3a/T--Northern_BC--Table_15.png)

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.

![](https://static.igem.org/mediawiki/2019/8/86/T--Northern_BC--Table_16.png)

Results: Minipreps look good. Proceed to screen for successful elimination of the XbaI (NEB Cat# R0145S) site by restriction enzyme digest.

![](https://static.igem.org/mediawiki/2019/a/a6/T--Northern_BC--Table_17.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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.

![](https://static.igem.org/mediawiki/2019/b/b1/T--Northern_BC--Table_18.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Experiments#transformation) 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.

![](https://static.igem.org/mediawiki/2019/a/af/T--Northern_BC--Table_19.png)

**July 17:**   *Jasneek, Michael*
PCR gene inserts.

![](https://static.igem.org/mediawiki/2019/5/58/T--Northern_BC--Table_20.png)

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.

![](https://static.igem.org/mediawiki/2019/8/82/T--Northern_BC--Table_21.png)

Set up the following digests:

![](https://static.igem.org/mediawiki/2019/d/d1/T--Northern_BC--Table_22.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose).
              •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

![](https://static.igem.org/mediawiki/2019/e/e2/T--Northern_BC--Table_23.png)

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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Agarose) 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.

![](https://static.igem.org/mediawiki/2019/2/2e/T--Northern_BC--Table_24.png)

**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.

![](https://static.igem.org/mediawiki/2019/7/77/T--Northern_BC--Table_25.png)

Ligate gGEM1 in to pGEM13 in a 3:1 molar ratio of insert to vector.

![](https://static.igem.org/mediawiki/2019/b/bb/T--Northern_BC--Table_26.png)

Left reaction on ice. Start: 1:30PM, End: 7:10PM
[Transform](https://2019.igem.org/Team%3ANorthern_BC/Experiments#transformation) 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](https://2019.igem.org/Team%3ANorthern_BC/Recipes#LB) and 25 [LB + Carb plates](https://2019.igem.org/Team%3ANorthern_BC/Recipes#Carb).

**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.

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# Team:Northern BC/Experiments

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TEAM

[Team Members](https://2019.igem.org/Team%3ANorthern_BC/Team)

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PROJECT

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[Design](https://2019.igem.org/Team%3ANorthern_BC/Design)

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WET LAB

[Notebook](https://2019.igem.org/Team%3ANorthern_BC/Notebook)

[Experiments](https://2019.igem.org/Team%3ANorthern_BC/Experiments)

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[Parts](https://2019.igem.org/Team%3ANorthern_BC/Parts)

[Results](https://2019.igem.org/Team%3ANorthern_BC/Results)

[Safety](https://2019.igem.org/Team%3ANorthern_BC/Safety)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3ANorthern_BC/Human_Practices)

[Public Engagement](https://2019.igem.org/Team%3ANorthern_BC/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Northern_BC)

# Northern BC iGEM 2019 Protocols

### Site-Directed Mutagenesis

**Primer Design:**

Design primers that are 30 - 45 base pairs in length, depending on the number of bases to be changed. Centering the primers over the position to be changed works best, however the primers could be offset to avoid formation of primer dimers. We haven’t found this to be a problem and tend to center our primers.

It is helpful to introduce a restriction site, if possible, so colonies can be screened by restriction enzyme digest rather than by sequencing. This saves time and money, although the final candidate will still have to be sequenced to confirm the correct mutation has been made without the introduction of additional mutations. The restriction enzyme site would have to be unique; that is, not present anywhere else in the plasmid.

**Template Plasmid:**

Smaller plasmids are generally easier to work with, although we have had success with larger plasmids (~ 8 kbp). Plasmids such as pUC18 or pUC19 work well for site-directed mutagenesis. Mini-prep the template plasmid from an overnight liquid culture.

**PCR Reaction and Conditions:**

Add the following components in this order. Keep the DNA Polymerase on ice at all times.

![Table of PCR Reagents](https://static.igem.org/mediawiki/2019/6/6f/T--Northern_BC--Protocols_Table.png)

A different DNA Polymerase may be used, but it must be high fidelity to avoid introducing random mutations. Taq DNA Polymerase should not be used for this purpose. We have been using Phusion® High-Fidelity DNA Polymerase *(NEB Cat# M0530)*.

**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)
              \*The extension temperature should be set according to the manufacturer’s recommendations for your DNA polymerase
              Repeat stage two for 18 cycles
Stage 3: 72 °C 20 minutes (final extension to catch any strands that were not fully extended earlier)
Stage 4: 4 °C indefinitely

Setting the program to end with an indefinite 4 °C period allows one to leave the reaction run over night. Leaving it at 4 °C in the thermocycler is no different from leaving it in the fridge. Running the PCR overnight means the reaction will be ready to work with first thing the following morning.

**DpnI Digest:**
There is now mixture of plasmid carrying the mutation and the original parental wild type DNA, so the parental DNA will need to be removed. Since bacteria methylate their DNA, the parental DNA will be methylated while all PCR-generated DNA will not. DpnI is a restriction enzyme that recognizes and cleaves a consensus sequence only when the DNA is methylated. Thus treatment with DpnI will degrade all wild type DNA, leaving only the PCR-generated mutant.

Add 1 uL of DpnI (NEB Cat# R0176S) directly to the reaction and incubate at 37 °C for at least two hours, but not more than 16 hours. We generally find three to four hours to be sufficient.

**Precipitate DNA to Concentrate and Clean-up:**
To your PCR reaction add 50 uL dH2O, 5 uL 5 M NaCl,1 uL 20 mg/mL glycogen, and 1 mL cold 100% ethanol.

Spin in a microcentrifuge at 13,000 rpm at 4 °C for at least 10 minutes, but not more than 30 minutes. Ideally, DNA should pellet at the tip of the tube, but it should not be packed so tight that it is hard to resuspend.

Pour off the ethanol and gently add 1 mL cold 70% ethanol to wash the pellet (do not resuspend the pellet).

Spin again as above, but for only five minutes.

Remove all ethanol and air dry on the bench for approximately 10 - 15 minutes. Do not let this go for too long as a pellet that is over dried seems to be harder to resuspend, affecting the transformation efficiency.

Transform the whole reaction into *E. coli* (DH5𝝰). We tend to resuspend the DNA with 50 uL of cells and carry on directly with the transformation protocol, but it could instead be resuspended in 5 uL of dH2O and then added directly to the cells.

**Troubleshooting:**
Presence of wildtype transformants this could be because the parental DNA is not efficiently methylated. Grow the cells in which the plasmid has been propagated for 24 hours to make sure the DNA is fully methylated before your mini-prep. You could also try using less template in the PCR or increasing the number of cycles in stage 2 of the program to 30.

### Bacterial Transformation

Add 5 - 10 uL of ligation reaction or up to 25 ng of circular plasmid DNA to 50 uL of RbCl2 competent cells, making an effort not to use a volume exceeding 5% of the volume of cells used.

**Note:** Use DH5𝝰 for plasmid storage and/or propagation and BL21, Rosetta, or other expression strain for protein expression and purification from a bacterial host.

**Controls:** Set up a negative and a positive control to assess cell competency

              **(+) Control:** 1 - 10 ng of supercoiled plasmid DNA (pUC19 works well) expressing the appropriate selective marker
              This should generate a ‘lawn’ of colonies - too many to count

              **(-) Control:** cells with no DNA
              Since the reactions will be plated on selective media, any cells that have not taken up the plasmid conferring resistance to
              the chosen antibiotic will not survive. Thus, there should be no growth on this plate.

Store all tubes on ice for 30 minutes.

Heat shock each reaction in a water bath for 90 seconds at 42 °C. The cells must be brought to temperature at an appropriate rate; thus, a water bath is most suitable for this purpose. The length of incubation is a function of the thickness of the microcentrifuge tube wall. If you find your transformations are not working well you can vary the incubation time. Most protocols call for an incubation time of anywhere from 30 seconds to two minutes, but 90 seconds has always worked well for us.

Without shaking the tubes, gently transfer them directly to ice and allow the cells to cool for two minutes.

Add 100 uL of liquid LB (no antibiotic) and incubate on a gently shaking orbital shaker for 30 - 45 minutes at 37 °C. If a shaker is not available place the tubes in a 37 °C incubator or water bath and periodically and gently agitate them. This is a recovery phase in which the cells are healing after heat shock. You must be gentle with them and provide enough time for them to begin producing the antibiotic resistance selective marker expressed from the plasmid before plating on selective media.

Working near a flame and using aseptic techniques, plate the entire 100 uL transformation onto a selective plate. We like to pre-incubate our plates at 37 °C to avoid shocking the cells again. Pipette the aliquot to the center of the plate and then add four to five sterile glass plating beads. Cover the plate with the petri dish lid and gentle roll the beads to spread the cells over the entire surface of the plate. Pour the used beads into a weak bleach solution for cleaning, sterilization and re-use.
Incubate plates overnight (approximately 16 hours) at 37 °C. Do not leave plates longer than 16 - 20 hours. Plates can be stored at 4 °C until one is ready to work with a colony.

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# Team:Northern BC/Results

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TEAM

[Team Members](https://2019.igem.org/Team%3ANorthern_BC/Team)

[Attributions](https://2019.igem.org/Team%3ANorthern_BC/Attributions)

[Sponsors](https://2019.igem.org/Team%3ANorthern_BC/Sponsors)

PROJECT

[Description](https://2019.igem.org/Team%3ANorthern_BC/Description)

[Design](https://2019.igem.org/Team%3ANorthern_BC/Design)

[Collaborations](https://2019.igem.org/Team%3ANorthern_BC/Collaborations)

WET LAB

[Notebook](https://2019.igem.org/Team%3ANorthern_BC/Notebook)

[Experiments](https://2019.igem.org/Team%3ANorthern_BC/Experiments)

[Recipes](https://2019.igem.org/Team%3ANorthern_BC/Recipes)

[Parts](https://2019.igem.org/Team%3ANorthern_BC/Parts)

[Results](https://2019.igem.org/Team%3ANorthern_BC/Results)

[Safety](https://2019.igem.org/Team%3ANorthern_BC/Safety)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3ANorthern_BC/Human_Practices)

[Public Engagement](https://2019.igem.org/Team%3ANorthern_BC/Public_Engagement)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Northern_BC)

# Results

### Build RFC-10 Compatible Yeast Shuttle Vectors

We have chosen to build our opioid sensor in the yeast *Saccharomyces cerevisiae* in order to take advantage of already existing intracellular machinery that we intend to hijack for amplification of the signal that is generated when an opioid molecule binds our detector protein. In order to successfully express our biosensor components, we required several yeast shuttle vectors that were able to be propagated in *E. coli*, but our sensor components expressed in *S. cerevisiae*. These were very generously donated to us by **Dr. Stephen Rader** (UNBC), along with the yeast strain BY4741.

BY4741 is an S288C-derived laboratory strain of *S. cerevisiae* in which there is a partial or complete deletion of the open reading frame of various genes required to produce certain amino acids (Table 1). In order for the yeast to survive, they must be supplemented with these amino acids in their growth media. Alternatively, the gene for specific enzymes that are required to produce the amino acid can be incorporated into a yeast vector to act as an auxotrophic marker. Expressing our sensor components from shuttle vectors that also express these auxotrophic markers allows us to track uptake of our genes by yeast cells grown in media that lacks these amino acids. The auxotrophic markers we chose to use were HIS3 and LEU2, expressed from pRS423 and pRS425, respectively. Both of these shuttle vectors contain a gene whose expression confers resistance to Ampicillin to allow for selective propagation of these vectors in *E. coli* (Table 1).

![](https://static.igem.org/mediawiki/2019/9/97/T--Northern_BC--Results_1.png)

***Site-directed mutagenesis of the yeast shuttle vectors***

pRS423 and pRS425 are commonly used in laboratories as yeast shuttle vectors, and therefore, we chose to use these as a starting point to build our opioid biosensor. However, both of these vectors contain restriction enzyme sites in regions outside of the multiple cloning site that are part of the RFC-10 biobrick prefix and suffix (Figure 1). In order to make these vector RFC-10 compatible, we had to first remove these extra restriction enzyme sites, and we chose to do this by sequential site-direct mutagenesis. The first step was to design a forward and a reverse primer that overlapped the region we intended to mutate, with each of these containing a single base change that destroyed the restriction enzyme recognition sequence, but did not alter the amino acid coded for. We successfully removed both a PstI and XbaI site from pRS423, and an EcoRI and XbaI site from pRS425.

![](https://static.igem.org/mediawiki/2019/e/e1/T--Northern_BC--Results_2.png)

To verify our mutations were present and that no other mutations had been unintentionally made during our site-directed mutagenesis, we provided the Northern Analytical Laboratories Services Genetics Facility with our sample DNA and **Ms. Caitlin Pitt** kindly ran the sequencing experiments for us. Sequencing results confirmed that all mutations were indeed present. Going forward we named our new pRS423 vector ‘pGEM13’ and our new pRS425 vector ‘pGEM18’.

***Addition of the RFC-10-compatible biobrick multiple cloning site into the yeast shuttle vector***
In order to both insert our biosensor ‘detector’ gene into the pGEM13 backbone, as well as to create a yeast shuttle vector that was biobrick-compatible for parts insertion and characterization, we needed to add the biobrick prefix and suffix to the multiple cloning site of the shuttle vector. To do this, we ordered a fragment of DNA containing the biobrick prefix and suffix and inserted it into the EcoRI-SacI site of pGEM13 while at the same time maintaining as many of the shuttle vector multiple cloning sites as possible (Figure 2). The successful incorporation of the DNA fragment was confirmed by sequencing through the multiple cloning site with the M13 sequencing primers.

![](https://static.igem.org/mediawiki/2019/1/17/T--Northern_BC--Results_2_1.png)

### Effect of Methanol Addition on BY4741 Cell Growth

Since the opioid and non-opioid drug samples we intend to test in our system are all suspended in methanol by the chemical supplier, we had to make sure methanol was not going to kill our cells. To this end, we tested the effect of increasing amounts of methanol on the growth rate of BY4741 cells (Figure 3). We found that as long as the methanol concentration is kept below 1% we should not see considerable adverse effects when we test our biosensor with these drug samples.

![](https://static.igem.org/mediawiki/2019/2/2a/T--Northern_BC--Results_3_1.png)

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