iGEM 2018 — Team ULaval

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaval/Notebook

[Home](https://2018.igem.org/Team%3AULaval)

Project

[Description](https://2018.igem.org/Team%3AULaval/Description)
[Design](https://2018.igem.org/Team%3AULaval/Design)
[Results](https://2018.igem.org/Team%3AULaval/Results)[Interlab](https://2018.igem.org/Team%3AULaval/InterLab)

Parts

[Overview](https://2018.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaval/Basic_Part)

Notebook

[Lab Journal](https://2018.igem.org/Team%3AULaval/Notebook)
[Protocols](https://2018.igem.org/Team%3AULaval/Experiments)
[Achievements](https://2018.igem.org/Team%3AULaval/Achievements)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AULaval/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3AULaval/Public_Engagement)
[Safety](https://2018.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2018.igem.org/Team%3AULaval/Team)
[Attributions](https://2018.igem.org/Team%3AULaval/Attributions)
[Collaborations](https://2018.igem.org/Team%3AULaval/Collaborations)

Notebook

![](https://static.igem.org/mediawiki/2018/e/ef/T--ULaval--Notebook.svg)

June 4th – July 2nd

-gBlock reception and rescue

-Blunt-end cloning – No positives

![](https://static.igem.org/mediawiki/2018/a/a4/T--ULaval--labjournal1.png)

*Figure 1.* Typical result of colony PCR for gBlock rescue by blunt-end cloning. Positives should have a band at around 2Kb, higher than what is shown on gel (1Kb NEB DNA ladder). 192 colonies were tested.

July 2nd – September 10th

-gBlock rescue by PCR.

-gBlock amplification using primers with restriction sequence overhang.

-Sticky end cloning using PCR product – No positives

![](https://static.igem.org/mediawiki/2018/0/02/T--ULaval--labofigure2.jpg)

*Figure 2.* Initial tests for gBlock rescue using PCR. Initial tests were inconclusive.

![](https://static.igem.org/mediawiki/2018/3/32/T--ULaval--figure3labo.jpg)

*Figure 3.* Typical result of colony PCR for gBlock rescue by sticky-end cloning. Positives should have a band at around 2Kb, higher than what is shown on gel (1Kb NEB DNA ladder). 288 colonies were tested.

September 10th – October 16th

- Design and order of new primers for Gibson assembly.

- gBlock rescue by PCR using newly designed primers.

- Gibson assembly of rescued gBlocks.

- Confirmation and sequencing of obtained clones.

![](https://static.igem.org/mediawiki/2018/f/f0/T--ULaval--figure4labo.png)

*Figure 4.* gBlock rescue using newly designed primers. Wells 1-2: gBlock TH. Wells 3-4: gBlock DDC. Wells 5-6: gBlock DBH. Wells 7-8: gBlock PNMT.

![](https://static.igem.org/mediawiki/2018/8/89/T--ULaval--figure5labo.jpg)

*Figure 5.* Colony PCR following Gibson assembly and transformation. The presence of a band signifies successful insertion.

![](https://static.igem.org/mediawiki/2018/d/db/T--ULaval--figure6labo.jpg)

*Figure 6.* Confirmation of positive clones for gBlock insertion. Wells 1-4: gBlock DDC. Wells 5-10: gBlock DBH. Wells 11-16: gBlock PNMT. gBlock TH was confirmed on a separate gel later.

Positives were sequenced to confirm proper insertion and sequence quality. All positive cloned were sequence-confirmed.

igem@bcm.ulaval.ca


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaval/Experiments

[Home](https://2018.igem.org/Team%3AULaval)

Project

[Description](https://2018.igem.org/Team%3AULaval/Description)
[Design](https://2018.igem.org/Team%3AULaval/Design)
[Results](https://2018.igem.org/Team%3AULaval/Results)[Interlab](https://2018.igem.org/Team%3AULaval/InterLab)

Parts

[Overview](https://2018.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaval/Basic_Part)

Notebook

[Lab Journal](https://2018.igem.org/Team%3AULaval/Notebook)
[Protocols](https://2018.igem.org/Team%3AULaval/Experiments)
[Achievements](https://2018.igem.org/Team%3AULaval/Achievements)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AULaval/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3AULaval/Public_Engagement)
[Safety](https://2018.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2018.igem.org/Team%3AULaval/Team)
[Attributions](https://2018.igem.org/Team%3AULaval/Attributions)
[Collaborations](https://2018.igem.org/Team%3AULaval/Collaborations)

Protocols

Here we present all the protocols that we used or designed to realize our project.

Culture Media

**Culture media for bacteria (for 1L)**

**2YT+glucose (rich media)**
1% Yeast extract (10g)
0.2% Glucose (2g)
0.5% NaCl (5g)

**LB (complex media salt rich)**
0.5% Yeast extract (5g)
1.0% Tryptone (10g)
1.0% NaCl (10g)

**2LB-Lennox (Rich media)**
1% Yeast extract (5g)
2.0% Tryptone (20g)
0.5% NaCl (5g)

**LB-Lennox (Rich media)**
0.5% Yeast extract (5g)
1.0% Tryptone (10g)
0.5% NaCl (5g)

**Culture media for yeast (for 1L)**

**YPD**
1% Yeast extract (10g)
2% Tryptone (20g)
2% Glucose (20g)
For solid media, (about 20mL/petri dish 100x15mm), add 2% agar before autoclaving media.

**SC media**
0.174% Yeast nitrogen base without amino acids, without ammonium sulfate (1.74g)
2% Glucose (20g)
X Drop-out mix (according to the drop-out used)
Add a nitrogen source according to your experience and to what your media will contain (for example, antibiotics):

0.5% ammonium sulfate (5g)
or
0.1% monosodium glutamate (MSG) (1g)

For solid media (about 20mL/Petri dish 100x15 mm or 35mL/Omnitray), autoclave 2% agar (20g/L) and combine to the rest once everything cooled down to 55°C.

Bacteria Competent Cells (CaCl2 method)

**Methods:**

1. Start 2x5mL precultures of the desired cells and incubate overnight at 37°C.
2. The day after, start 2x500mL cultures of 2YT with 5mL of preculture.
3. Reach a DO (600nm) of 1/mL (do not overreach).
4. Centrifugate cells at 5000 RPM for 20 min at 4°C.
5. Perform all the coming steps very gently.
6. Resuspend cells in 0.1M CaCl2 (100mL/pellet).
7. Let 3h on ice. (Start to resuspend after 1h30).
8. Centrifugate at 5000 RPM for 20 min.
9. Resuspend pellet in 0.1M CaCl2 + 15% glycerol (25mL/pellet).
10. Aliquot in 2mL tubes with screw caps and conserve at -80°C.

**Notes:**

Perform everything on ice because cells are very fragile.
Keep pipets at -20°C and tubes at -80°C the day before the experiment.
Sterilize centrifuge bottles.
Prepare 250mL of 0.1M CaCl2 and 100mL of 0.1M + 15% glycerol. Sterilize. Conserve at 4°C and transfer at -20°C 30 min before use.

Colony PCR (Bacteria)

Colony PCR works best with small fragments (200-500bp)
**Methods:**

1. Add 40µL of sterile water or 20nM NaOH in a PCR tube.
2. Touch gently the colony to test with a tip and resuspend in water (or NaOH).
3. Incubate 20 min at 95°C in the thermocycler.
4. Centrifuge 5 min at 4000 RPM.
5. Prepare the mastermix:

|  | One reaction (µL) |
| --- | --- |
| Sterile Water | 17.35 |
| Bioshop PCR Buffer [10X] | 2.5 |
| dNTP mix [10mM] | 0.5 |
| MgCL2 [25mM] | 1.5 |
| Reverse Primer [10µM] | 0.5 |
| Forward Primer [10µM] | 0.5 |
| Bioshop Taq DNA Polymerase [5U/µl] | 0.15 |
| Final volume | 23 |

6. Transfer 2µL of supernatant in a new PCR tube in addition of the PCR mix.

**Conditions:**

5 min at 94°C

**35 cycles of:**
30 s at 94°C
30 s at 55°C (or the appropriate Tm of your oligos)
X min at 72°C (Time according to the length of your PCR product, 1 min/kb)
3 min at 72°C
Hold at 10°C

Colony PCR (Yeast)

Colony PCR works best with small fragments (200-500bp)
**Methods:**

1. Put 15µL of sterile water in a PCR tube.
2. Touch slightly the colony to test with a tip and resuspend in water.
3. Incubate in the PCR machine for 20 min at 95℃.
4. Centrifuge 4 min at 15000 RPM.
5. Prepare the mastermix:

   |  | One reaction (µL) |
   | --- | --- |
   | Sterile Water | 16.85 |
   | Bioshop PCR Buffer [10X] | 2.5 |
   | dNTP mix [10mM] | 0.5 |
   | MgCL2 [25mM] | 1.5 |
   | Reverse Primer [10µM] | 0.5 |
   | Forward Primer [10µM] | 0.5 |
   | Bioshop Taq DNA Polymerase [5U/µl] | 0.15 |
   | Final volume | 22.5 |
6. Transfer 2.5µL of supernatant in a new PCR tube containing the PCR mix.

**Conditions:**

5 min at 94°C

**35 cycles of:**
30 s at 94°C
30 s at 55°C (or the appropriate Tm of your oligos)
X min at 72°C (Time according to the length of your PCR product, 1 min/kb)
3 min at 72°C
Hold at 10°C

Competent Cells (Yeast)

**Methods:**

1. 5mL preculture overnight
2. Yeast cells inoculated from fresh preculture and grown overnight until OD 0.5-0.7 in YPD (100mL culture).
3. Split 50mL culture in Falcon tubes. Harvest cells by centrifuging at 500g for 5 min at RT (~1500 RPM).
4. Wash with 5mL sterile water.
5. Centrifuge at 500g for 5 min.
6. Wash with 5mL sterile SORB. Centrifuge again.
7. Remove SORB with aspiration (sterile tips!).
8. Resuspend cells in 360μL SORB and 40μL carrier DNA (salmon sperm DNA 10mg/mL - boiled for 5 min and cooled on ice) per 50mL culture.
9. Aliquot (20µL) and place in freezer (-80°C).

|  | Stock solution | SORB 500mL | SORB 250mL |
| --- | --- | --- | --- |
| 100mM LiOAc | 1M | 50 | 25 |
| 10mM Tris pH 8.0 | 1M | 5mL | 2.5mL |
| 1mM EDTA | 1M | 1mL | 0.5mL |
| 1M sorbitol | 182.17g/mol | 91.0g | 45.5g |

Dissolve sorbitol in ~100mL water with stir bar on plate. Complete with above solutions and bring up to desired volume. Filter sterilize.

Gibson Cloning

Gibson cloning is used to clone one or more fragments in a vector of interest. In the lab, we generally use PCR amplified vector and fragments. Here is the general procedure in that specific case.

**Amplification of vector:**

Use a high fidelity polymerase (like the Kapa HiFi hotstart polymerase) to amplify your vector of interest. Your oligos should be around 20-24 nt (it depends on the Tm of the oligo - see following section) and should be designed as to amplify your vector from each side of the insertion site. PCR reaction must then be digested with DpnI to get rid of parental plasmid. Purify on column. A vector can also be digested and directly used for cloning. In that case, make a double-digestion and make sure it is complete.

**Amplification of fragments (inserts):**

Use a high fidelity polymerase (like the Kapa HiFi hotstart polymerase) to amplify your fragments of interest (from gDNA or plasmid). Design a forward primer with an overlap region (≥ 16 nt - usually 20 to 24 nt) at the 5´-end, which is complementary to the reverse primer of the vector. The 3´-end of the forward primer (around 20 nt - it depends on the Tm, try to have a 50% GC content) should contain insert-specific sequences to amplify the target fragment. The “≥ 16 nt” overlap region of the forward primer should be complementary to the 5´-end sequence of the vector which corresponds to the insertion site. The size of the overlap region is determined by the number of nucleotides needed to reach a Tm > 48°C. If necessary, one can also add nucleotides between the overlap region and gene-specific sequence region to ensure, for example, that the expressed protein is in frame. Similarly, the reverse primer should also contain an overlap region (≥ 16 nt - usually 20 to 24 nt) at the 5'-end and an insert-specific sequence at the 3'-end.
If the amplification is made from a plasmid, digest the PCR reaction with DpnI and purify on column.

**Manufacture Gibson mix:**

1. Prepare 6mL of 5X ISO Buffer in a 15mL falcon tube as follows:

3mL 1M Tris-HCl pH 7.5

+ 150µL 2M MgCl2
+ 240µL 100 mM dNTP mix (25mM each: dGTP, dCTP, dATP, dTTP)
+ 300µL 1M DTT
+ 1.5g PEG-8000
+ 300µL 100mM NAD
+ dH20 to 6mL
Store at -20°C in 320µL aliquots.

2. Prepare 1.2mL of Gibson assembly master mix as follows:

320µL 5X ISO Buffer

+ 0.64µL 10U/µL T5 exonuclease (This is optimized for 20-150 bp sequence homology overlaps)
+ 20µL 2U/µL Phusion polymerase
+ 160µl 40U/µL Taq ligase
+ 700µL dH20
Store at -20°C in 7.5µL aliquots in PCR tubes (or strips).

**Gibson cloning:**

1. Thaw a 7.5µL aliquot of the Gibson assembly master mix, and keep on ice until use.
2. Measure the DNA concentration (ng/µL) of each assembly piece (on an agarose gel).
3. Add 50ng of the vector backbone (PCR or linearized) and a 1:2 (vector:insert) molar ratio amounts of the other assembly pieces to the thawed 7.5µL master mix in a 10µL total volume assembly reaction mixture as follows:

vector backbone (50ng)

each insert (to a 1:2 molar ratio with backbone)
7.5µL Gibson assembly master mix
ddH20 to 10µL
The vector:insert ratio must be adapted to the size of the fragments to clone. For smaller fragments, more inserts should be put in the reaction.

4. Incubate the assembly reaction at 50°C for 60 min, and then place on ice.
5. Transform 5µL of the assembly reaction into 100µL of competent *E.coli* and plate 100µL and 900µL on 2YT plates with the appropriate antibiotic(s).

Gibson cloning is hard to achieve with repeated sequences.
See the original brochure for more information about the general protocol and the procedure to design the fragments for assembly: File:Manual from NEB for Gibson cloning.pdf

Transformation of Competent Bacteria

**Methods:**

1. Transfer 0.1mL of competent cells in a 15mL tube cooled on ice.
2. Add 2-5µL of plasmid.
3. Incubate 15 min on ice.
4. Perform a heat shock at 42°C for 1 min.
5. Put on ice for 5 min.
6. Add 900µL of 2YT media without antibiotics.
7. Incubate 45 min at 37°C with agitation.
8. Spread 100µL of cells on 2YT+glucose+antibiotic media.
9. Centrifuge the remaining volume (900µL), resuspend pellet in 100µL then spread.
10. Incubate overnight at 37°C.

Transformation of Competent Yeast

**Methods:**

1. 8μL of DNA (0.5-1μg/μL) and 20μL of thawed competent cells are placed into sterile tube (or PCR tubes in strips or plates if many transformations are done) and mixed.
2. 100 μL of Plate mixture is added.
3. Cells are mixed thoroughly and incubated at room temperature for approximately 30 min (without shaking).
4. 15μL DMSO is added.
5. The cells are placed at 42°C for 15-20 min in a water bath without shaking.
6. Centrifuge at 2000 RPM for 3 min and aspirate the supernatant.
7. Add 100μL YPD and incubate at 30°C for at least 2 hours (4 hours minimum in the case of a genomic integration) in a water bath without shaking.
8. The cells are plated on selective medium. In the case of multiple transformations, cells can be plated in a well of a 6-well plate previously filled with 4mL of media with agar. Make sure that all the liquid is well absorbed before removing the glass beads.
9. Place plates upside down in incubator at 30°C for 2–3 days.

If PCR plates are used, use aluminium seal to keep it sterile.
PLATE Mixture (combine all solutions previously autoclaved):

|  |  |
| --- | --- |
| PEG 3350 45% sterile | 90mL (final concentration 40%) |
| Li OAc 1M | 10mL (final concentration 100mM) |
| Tris-HCl (pH 7.5) 1M | 1mL (final concentration 10mM) |
| EDTA 0.5M | 0.2mL (final concentration 1mM) |

HPLC detection of standards and metabolites

**Solutions to prepare:**
Solvent : 0.1M HCL

1. Prepare a 1L bottle with 990mL of ultra pure water.
2. Add 10mL of 10M HCl (37%).
3. Stir gently. Strong acid solutions are hot: be careful and wear glasses and gloves.

Stock solutions of all standards:

1. Weight 100mg of standard powder.
2. Put in a 10mL volumetric flask.
3. Let the powder dissolve in solvent (don’t fill until the flask gauge). Mix by inversion.
4. Sonicate to help dissolution and remove bubbles.
5. Fill the flask to reach the gauge.
6. Filter with 0.2μm filter.

Working solution of all standards:

1. Perform serial dilutions of all standards.
2. Use well calibrated micropipettes or volumetric pipettes.

![](https://static.igem.org/mediawiki/2018/2/28/T--ULaval--figureHPLC.png)

*Figure 1:* Serial dilution of the standards

**HPLC programmation:**

UV detector: 210nm
Mobile phase: Water/MeCN3/TFA: 90/10/0.1
Debit: 0.5mL/min
End of cycle: 10 min
Injection volume : see *Figure 1*

**Mix of all standards :**

1. Prepare a 10mL flask containing 5mL of solvent (HCl 0.1M).
2. Add 500μL of each standard (tyrosine, adrenaline, DOPA, L-DOPA) from working solutions (100ng/μL).
3. Fill the flask to reach the gauge.
4. Inject 10μL of the mix in the HPLC.

**Detection of metabolites and standards:**
Extraction from intracellular content:

1. Prepare 100mL of PBS 1X at pH 7.4 in a 250mL bottle.
2. Prepare a 5mL solution of zymolyase 70U/mL.
3. Centrifuge 1mL of 72 hours yeast culture in an eppendorf tube at 10000 RPM for 5 min.
    Transfer 500μL of supernatant in a new eppendorf tube for extracellular content analysis.
4. Resuspend cells with 1mL of PBS 1X pH 7.4.
5. Centrifuge again at 3000RCF for 5 min. Discard supernatant.
6. Resuspend in 500μL of PBS1x pH7.4 containing 35U of zymolyase.
7. Incubate 1 hour at 37°C. Yeast cells become spheroplasts during that step.
8. After incubation, add 500μL of acetonitrile in order to break cells and precipitate proteins.
9. Vortex for 1 min then centrifuge at maximum speed for 1 min.
10. Extract supernatant while avoiding the pellet. With the help of a syringe and a 0.2μm filter: filter supernatant and transfer in a new eppendorf tube.
11. Concentrate 500μL of supernatant with nitrogen gaz: let dry then resuspend in 100μL of acetonitrile.

 Extracellular content:

1. Add 250μL of 2M HClO4 to the supernatant.
2. Centrifuge 10 min at 13000 RPM.
3. Extract supernatant while avoiding the pellet. With the help of a syringe and a 0.2μm filter: filter supernatant and transfer in a new eppendorf tube.
4. Conserve supernatant until injection in the HPLC.

igem@bcm.ulaval.ca

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:ULaval/Results

[Home](https://2018.igem.org/Team%3AULaval)

Project

[Description](https://2018.igem.org/Team%3AULaval/Description)
[Design](https://2018.igem.org/Team%3AULaval/Design)
[Results](https://2018.igem.org/Team%3AULaval/Results)[Interlab](https://2018.igem.org/Team%3AULaval/InterLab)

Parts

[Overview](https://2018.igem.org/Team%3AULaval/Parts)
[Basic Parts](https://2018.igem.org/Team%3AULaval/Basic_Part)

Notebook

[Lab Journal](https://2018.igem.org/Team%3AULaval/Notebook)
[Protocols](https://2018.igem.org/Team%3AULaval/Experiments)
[Achievements](https://2018.igem.org/Team%3AULaval/Achievements)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AULaval/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3AULaval/Public_Engagement)
[Safety](https://2018.igem.org/Team%3AULaval/Safety)

Team

[Team members](https://2018.igem.org/Team%3AULaval/Team)
[Attributions](https://2018.igem.org/Team%3AULaval/Attributions)
[Collaborations](https://2018.igem.org/Team%3AULaval/Collaborations)

Results

![](https://static.igem.org/mediawiki/2018/e/ef/T--ULaval--Notebook.svg)

**Introduction**

For our project AdrenaYeast, we planned on producing three different plasmids harboring different enzymes that allowed for the biosynthesis of adrenaline and some metabolic intermediates in the yeast *S. cerevisiae*. We, however, encountered many problems in the first weeks of work. As this is our first iGEM participation, we struggled to secure access to lab facilities at certain times. There were also some communication problems that resulted in some work being done in duplicate, or not at all. In the end, we did not manage to produce all the desired plasmids shown in *Figure 1*, but we were able to clone all our parts in the desired initial vectors, and to sequence confirm them. Here are the main steps that we worked through to produce the desired plasmids, and the steps we planned on doing to finish the plasmid and strain construction.

![](https://static.igem.org/mediawiki/2018/8/8e/T--ULaval--figureAResults.png)

*Figure 1*. A) Produced plasmids B) Planned plasmids

**Planning and DNA Preparation**

First, we started by designing our parts. We did a literature review to assess the relative activity of the enzymes, to verify that they did not necessitate post-translational modifications (PTM) and to check which isoform was active in our chassis’ conditions. We also used the IDT optimization algorithm to codon-optimize the sequences for use in our chassis. After identifying the proper isoforms and assessing that the parts would likely be active and non-toxic, we chose yeast promoters and terminators from the iGEM registry to add to our parts and create our own parts. We managed to generate four parts using different promoter, coding sequence (CDS) and terminator combinations, all under 3Kb in length. This step was crucial, since we wanted to order the designed parts as gBlocks from IDT. This would allow us to skip part assembly and go directly to plasmid assembly. After a few synthesis problems and some sequence optimization to allow for proper synthesis, we received our gBlocks from IDT. Here, we encountered another problem. One of the students working in the laboratory did not use the proper solution to resuspend some of the gBlocks, meaning that we lost most of the DNA present in the tube. The *Figure 2* shows a gel electrophoresis on 1% agarose of 2μL of the concentrated gBlocks. We can see that the gBlocks for LDDC and PNMT are much less concentrated than the other two.

![](https://static.igem.org/mediawiki/2018/a/a7/T--ULaval--figureBResults.png)

*Figure 2*. gBlock rescue and migration to confirm quality

**Blunt-End Cloning**

Initially, we tried to rescue gBlocks by blunt-end cloning in a linearized blunt-end vector, as we ordered our gBlocks 5’ phosphorylated. Sadly, after many tries, we did not manage to get a single positive clone for gBlock insertion.

**Restriction enzymes cloning**

We then turned ourselves to directional restriction enzyme cloning. We designed and ordered primers with overhang that contained restriction sites to amplify our gBlocks with. Sadly, doing a PCR on gBlocks of above 1Kb is not recommended by IDT, and we had very low amplification efficiency, leading to very low cloning efficiency. Furthermore, restriction cloning necessitated double digestions and at least two rounds of column purification, meaning that we lost most of our DNA by the end of the process. We did not manage to get any positives using restriction enzyme cloning. As the Jamboree was approaching, we entrusted the remaining cloning and troubleshooting to a single person, hoping it would speed up the process.

**Gibson Assembly**

It was decided to continue using the Gibson assembly technique, as restriction cloning was inefficient. New primers for Gibson assembly were carefully designed. Gibson cloning was carried out with the goal of inserting each gBlock individually in either one of the available backbones, each carrying either a resistance to hygromicin or nourseotricin, pRS31H and pRS31N respectively. Using Gibson assembly and the new primers, we were able to generate the plasmids showed in *Figure 1A* with high efficiency (41/48 colonies tested were positive for gBlock insertion compared to 0/480 for other cloning methods). Surprisingly, we were only able to insert the fragments in pRS31N. The *Figure 3* below shows, for all genes-gBlocks, the linearized plasmid pRS31N, the amplified composite gBlock and finally the linearized plasmid containing the amplified gBlock. The next step would be to design new Gibson assembly primers and carry out cloning to combine two gBlocks into one plasmid. Moreover, it would also be necessary to construct these plasmids in the pRS31H backbone to be able to co-transform both plasmids in a single yeast strain.

![](https://static.igem.org/mediawiki/2018/6/6c/T--ULaval--figure3Results.png)

*Figure 3*. Confirmation of gBlocks inserts in pRS31N

**HPLC**

Sadly, we were not able to characterize our parts properly. In order to do so, we ordered a HPLC reverse-phase analytical column made by SIELC Technologies for the efficient separation of catecholamines. We wanted to use it to purify our different products and thus verify the activity and expression of our enzymes. As they are not natively present in our chassis, the detection of their reaction products would have been a confirmation of the enzyme’s expression and activity. After waiting for a month to receive the column, we learned that it was lost in the mail and that we would not receive it in time for proper part characterization.

igem@bcm.ulaval.ca