iGEM 2021 — Team ULaval

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# Team:ULaval/Notebook

Project
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[Project Description](https://2021.igem.org/Team%3AULaval/Description)
[Experiments](https://2021.igem.org/Team%3AULaval/Experiments)
[Parts](https://2021.igem.org/Team%3AULaval/Parts)
[Results](https://2021.igem.org/Team%3AULaval/Results)
[Safety](https://2021.igem.org/Team%3AULaval/Safety)
[Project Modelling](https://2021.igem.org/Team%3AULaval/Model)
[Engineering Success](https://2021.igem.org/Team%3AULaval/Engineering)
[Proof of Concept](https://2021.igem.org/Team%3AULaval/Proof_Of_Concept)
[Syrup Microbiology](https://2021.igem.org/Team%3AULaval/Syrup_Microbiology)

Human Practices
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[Human Practices](https://2021.igem.org/Team%3AULaval/Human_Practices)
[Implementation](https://2021.igem.org/Team%3AULaval/Implementation)

Team
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[Team](https://2021.igem.org/Team%3AULaval/Team)
[Attributions](https://2021.igem.org/Team%3AULaval/Attributions)
[Notebook](https://2021.igem.org/Team%3AULaval/Notebook)

Collaboration & Communication
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[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

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Project
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[Project Description](https://2021.igem.org/Team%3AULaval/Description)

The Basics
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Experiments](https://2021.igem.org/Team%3AULaval/Experiments)
[Parts](https://2021.igem.org/Team%3AULaval/Parts)
[Results](https://2021.igem.org/Team%3AULaval/Results)
[Safety](https://2021.igem.org/Team%3AULaval/Safety)

What Makes It Great
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Project Modelling](https://2021.igem.org/Team%3AULaval/Model)
[Engineering Success](https://2021.igem.org/Team%3AULaval/Engineering)
[Proof of Concept](https://2021.igem.org/Team%3AULaval/Proof_Of_Concept)
[Syrup Microbiology](https://2021.igem.org/Team%3AULaval/Syrup_Microbiology)

Human Practices
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Human Practices](https://2021.igem.org/Team%3AULaval/Human_Practices)
[Implementation](https://2021.igem.org/Team%3AULaval/Implementation)

Team
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Team](https://2021.igem.org/Team%3AULaval/Team)
[Attributions](https://2021.igem.org/Team%3AULaval/Attributions)
[Notebook](https://2021.igem.org/Team%3AULaval/Notebook)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

# Notebook

# This page summarizes the timeline of our project this year. As you can see we formed our team in January, discussed multiple project ideas in February, and chose our first project idea in March, that changed along the way until July, when we chose our final project idea. From there, we contacted professors in our university to help us with the science aspect of our project until mid-October. From there, we focused on the deliverables for iGEM such as the wiki and the 20-minute presentation.

February 2021

February 2nd, 2021     Integration meeting with members

February 13th, 2021    Meeting to plan out lab work

February 18th, 2021    Beginning of financial planification

February 19th, 2021    Beginning of wet lab planification

February 21st, 2021    Planification for fundraising, its video and bonuses for people who help us out (including a cookbook)

March 2021

March 4th, 2021      Contact with Maman Veut Savoir for a science communication collaboration New project logo!

March 16th, 2021     Advancement of the ethical approuval of our survey for consummers (started last year)

March 23rd, 2021    Beginning of brainstorming process for our promotional video

March 26th, 2021    Planification of our dextran order (quantity, size, etc)

March 30th, 2021    Beginning of a collaboration with passionbouffe for our cookbook

April 2021

April 1st, 2021      Beginning of sponsorship requests

April 8th, 2021     Contacting people for our Human practices

April 15th, 2021   Meeting with the PPAQ to discuss our project and its future applications / Team registration / Our project is selected for an interview with *Forces Avenir*, a provincial entrepreneurship contest

April 19th, 2021     Beginning of our wet lab

May 2021

May 5th, 2021      Fundraising video is ready! / Beginning of our wet lab!!

May 19th, 2021    First contact with iGEM Concordia for a collaboration

May 25th, 2021    iGEM Dusseldorf contacts us for a postcard collaboration

June 2021

June 1st, 2021       Practice for our presentation for the *Forces Avenir* interview
/ Our survey for consummers is approved!!
/ Beginning of the brainstorm for our wiki's layout
/ New project : Determine the microorganisms present in ropy maple syrup and normal maple syrup

June 7th, 2021     Beginning of our modelling planification

June 12th, 2021    Meeting with Sébastien Houde for our project modelling

June 16th, 2021    We started planning our team promotion video
/ We started sharing our consummer survey

July 2021

July 6th, 2021        Meeting with Hossein to talk about modelling

July 13th, 2021       Planification for our radio interview

July 14th, 2021     Radio interview with CKRL, a local city radio

July 16th, 2021       Postcards for our contribution to Düsseldorf printed and sent

July 30 - 31, 2021  JulyGEM

August 2021

August 11th, 2021      Analysis of our survey to consummers

August 28th, 2021    Registration for the Giant Jamboree

September 2021

September 1st, 2021      We started writing our wiki

September 9th, 2021    We finally got our microbiology results and could identify the microorganisms present in ropy maple syrup

September 11th, 2021    Our first fall in person work meeting

September 13th, 2021    Planification of our meeting with the MAPAQ to discuss the legal aspects of our project

September 16th, 2021    Women in STEM - Diversity and Inclusion workshop!!
/ Anouncement of the Rossalind Chronicles

September 26th, 2021    End of our wet lab.

October 2021

October 6th, 2021      Gala *Forces Avenir*.

October 26th, 2021    Morrin Centre presentation.

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# Team:ULaval/Engineering

Project
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Project Description](https://2021.igem.org/Team%3AULaval/Description)
[Experiments](https://2021.igem.org/Team%3AULaval/Experiments)
[Parts](https://2021.igem.org/Team%3AULaval/Parts)
[Results](https://2021.igem.org/Team%3AULaval/Results)
[Safety](https://2021.igem.org/Team%3AULaval/Safety)
[Project Modelling](https://2021.igem.org/Team%3AULaval/Model)
[Engineering Success](https://2021.igem.org/Team%3AULaval/Engineering)
[Proof of Concept](https://2021.igem.org/Team%3AULaval/Proof_Of_Concept)
[Syrup Microbiology](https://2021.igem.org/Team%3AULaval/Syrup_Microbiology)

Human Practices
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Human Practices](https://2021.igem.org/Team%3AULaval/Human_Practices)
[Implementation](https://2021.igem.org/Team%3AULaval/Implementation)

Team
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Team](https://2021.igem.org/Team%3AULaval/Team)
[Attributions](https://2021.igem.org/Team%3AULaval/Attributions)
[Notebook](https://2021.igem.org/Team%3AULaval/Notebook)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

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Project
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Project Description](https://2021.igem.org/Team%3AULaval/Description)

The Basics
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Experiments](https://2021.igem.org/Team%3AULaval/Experiments)
[Parts](https://2021.igem.org/Team%3AULaval/Parts)
[Results](https://2021.igem.org/Team%3AULaval/Results)
[Safety](https://2021.igem.org/Team%3AULaval/Safety)

What Makes It Great
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Project Modelling](https://2021.igem.org/Team%3AULaval/Model)
[Engineering Success](https://2021.igem.org/Team%3AULaval/Engineering)
[Proof of Concept](https://2021.igem.org/Team%3AULaval/Proof_Of_Concept)
[Syrup Microbiology](https://2021.igem.org/Team%3AULaval/Syrup_Microbiology)

Human Practices
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Human Practices](https://2021.igem.org/Team%3AULaval/Human_Practices)
[Implementation](https://2021.igem.org/Team%3AULaval/Implementation)

Team
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Team](https://2021.igem.org/Team%3AULaval/Team)
[Attributions](https://2021.igem.org/Team%3AULaval/Attributions)
[Notebook](https://2021.igem.org/Team%3AULaval/Notebook)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

Engineering Success

![](https://static.igem.org/mediawiki/2021/f/f7/T--ULaval--cycleprod.png)
Figure 1: DBTL cycle used to summarize our project idea.

Since 2020, we have been working on a solution against maple syrup polymers (aSAP). To organize
our project, we used the Design-Build-Test-Learn cycle (DBTL cycle) proposed to us by one of our
mentors, Michel Guertin. This strategy follows four steps: (1) the Design phase is where we
define the problem, propose multiple solutions and identify the biological components to
construct; (2) the Build phase is where we synthesise, assemble and/or edit the components; (3)
the Test phase is where we characterise all the components we build to identify the ones that
work best and (4) the Learn phase that allows us to analyse all the data generated to start the
next DBTL cycle. Last year, we did not have access to the laboratories, so we concentrated all
of our energy into the design and build part of our project. This year, we put emphasis on the
build, test and learning phases to improve our solution. This section summarizes what we did
last year and what we have done this year.

![](https://static.igem.org/mediawiki/2021/f/f7/T--ULaval--cycleprod.png)

Figure 1: DBTL cycle used to summarize our project idea.

Design

Build

Test

Learn

Last year, one of our members, Benjamin Ouellet, suggested that we work on maple syrup as it
is an important part of our economy and our culture in Canada. After meeting and discussing
with experts in the field such as Marie Filteau, Luc Lagacé, Vincent Poisson and Jean-Michel
Lavoie, we decided to work on ropy syrup, a kind of maple syrup with a high concentration of
dextrans causing viscosity (Lagace and al., 2018). Since it is classified as a flavor defect in
Quebec’s
legislation, it needs to be destroyed (Légis Québec, 2012). This causes food waste, in addition to
energy and
economical losses for maple syrup producers.

These dextrans are exopolysaccharides produced by bacteria that come from the environment
and are introduced in the tubing system during maple sap collection (Lagace and al., 2018). These
dextrans then
cause maple syrup to have a ropy and viscous texture3. Up to now, there is no way to detect
if maple saps are contaminated with dextrans. Thus, the ropiness of the syrup causes damage
to expensive equipment.

The solution we developed to solve this problem is a treatment using an enzyme, a
dextranase, to degrade the dextrans polymers (Bashari and al., 2013). We then decided that it is best
for our treatment to be used after the evaporation process when the ropy syrup is already
produced, so only the ropy syrup gets the treatment and the rest of the maple syrup
production stays untouched. For the dextranase, there are specific conditions in which the
enzyme needs to be active: high viscosity, low temperature, and slightly acidic
pH (Lagace and al., 2018, Khalikova and al., 2005).

![](https://static.igem.org/mediawiki/2021/9/94/T--ULaval--processprod.png)

Figure 2: Maple syrup production cycle

Last year, our team was able to find one dextranase candidate expressed in Gelidibacter
algens. We choose this dextranase because its properties could fit with the
expected
experimental conditions in ropy maple syrup. Also, discussions with Marie-Ève Picard, a
member of a professor’s lab here at the university, helped us decide to use pET-28a as an
expression vector for both the dextranase. It is an expression vector for Escherichia coli
strains so we choose to use a codon-optimized sequence of the dextranase in E. coli
Furthermore, we looked through the literature to find previous experiments on expression and
purification of dextranases. We found that the dextranase of S.
mutans showed proteolysis
during those experiments (Kim and al., 2011) and that this modification enhanced its activity. The
authors
identified the protein segments to remove from the enzyme to gain activity. Using sequence
alignment, we were able to evaluate the segments we needed to remove from the G. algens.
dextranase and designed the part accordingly.

For further details regarding the design of our project, we refer you to our team wiki from
the 2020 competition.

As planned last year, we ordered our dextranase expression vectors through Twist
Biosciences. We transformed the pET28a vector containing the G.
algens’ dextranase coding
sequence in E. coli in DH5ɑ strain for stock conservation and
sequence verification. Once
the sequencing confirmed that the sequence corresponded to our design, the pET28a-G.algens
was transformed in E. coli BL21 strain to start the Test phase.

The first step to test our part was to successfully express the enzyme in our induction
system. To do so, we performed an experiment to optimize the expression of our protein by
testing multiple induction conditions. The samples that showed the largest expression of
protein were then sonicated to verify if the dextranase was soluble. By those experiments,
we showed that the dextranase of G. algens was best expressed
and showed higher solubility
under the following induction condition: 18 hours of induction with 1mM IPTG at 18C. Also,
after a quick purification test, we knew that the protein could be successfully purified. We
used these first purified samples to perform a Nelson-Somogyi assay allowing us to determine
if the dextranase was still active. We were thrilled to see that the enzyme was able to
degrade dextran even after the whole purification protocol. To our knowledge, it was the
first time the G. algens dextranase was successfully expressed
and purified.

The second step was to purify a large amount of our enzyme to do the enzymatic and
rheological assays. We used the determined optimized condition to perform the expression and
the purification of the dextranase. Indeed, we obtained a high concentration (636uM) of
purified protein in our 15mL sample. This was large enough for our needs so we moved
forward with the enzymatic assays.

For the enzymatic assays, we used a miniaturized version of the Nelson-Somogyi protocol
allowing us to use less dextranase for each reaction (Borkowska and al., 2019). Because no experimental
data were
available on the properties of this enzyme, we tested the activity and the stability of the
dextranase at various temperatures and pH on dextrans of 3 different molecular weights. We
found that the stability and activity are not affected by the range of pH that we
tested (5.5-7) and that the dextranase does not seem to have a preferred dextran weight for
its activity. However, the temperature seems to impact the activity and the stability of the
dextranase. Surprisingly, the optimal activity was observed at 40°C although the optimal
stability is measured at 18°C. We also tested our dextranase directly in the ropy syrup at
room temperature. We saw a decrease in the viscosity of the sample treated with the
dextranase compared to the control. This observation was only visual, so we wanted to add
some quantitative data to prove the efficiency of our treatment.

We used a rheometer to collect the quantitative data on our dextranase treatment. We
measured the viscosity of a ropy maple syrup sample and compared it to the treated ropy
syrup at 20°C. It showed a decrease in viscosity, however, this decrease was found very
similar to the one caused only by the dilution of the ropy syrup with the buffer. We know
that our enzymatic treatment will be applied at a lower temperature so we also tested the
effect of the dextranase at 10°C. The treatment caused a better relative decay of viscosity
at this temperature. It is explained by the fact that the sample viscosity increases
exponentially with the diminution of the sample temperature.

From our analysed data, we conclude that our expression vector and strategy worked
efficiently. Also, the approach for protein purification was successful. However, the
properties of the G. algens dextranase were not what we expected. Indeed, we expected that
its activity would be maximal at a temperature around 10-15°C, but we showed that it was
around 40°C. Therefore, the dextranase treatment is not at its maximal potential at the
conservation temperature of maple syrup. On the other hand, we learned that maple syrup with
food additives would never be sold as maple syrup. We were already reflecting on the
possibility of producing other sugar bases with the product of our treated ropy syrup. Thus,
there is no relevance in keeping the enzymatic treatment at a low temperature(preservation
of maple syrup). It is very convenient because our enzyme is more active at higher
temperatures.

Due to time constraints, we were not able to perform all rheological tests we wanted. So
there is a lack of information about the impact of the dextranase on the ropy syrup for
longer incubation time and for wider temperature range. Nevertheless, we saw that the G.
algens dextranase is active and accomplishes its purpose. We also observed that the dilution
factor induced when we add the treatment to the syrup is quite high and it makes it
difficult to measure the treatment effect at room temperature. To improve the treatment, we
could concentrate the enzyme to add the same quantity of dextranase without diluting the
sample as much.

In Short:

We produced a lot of data in the Test phase demonstrating that our strategy worked:

* We were able to express and purify the dextranase of G.
  algens.
* The dextranase is active and degrades dextrans.
* Our treatment decreases the viscosity of ropy syrup in small samples.

However, it could be improved in many ways:

* We have to do more exhaustive rheological tests to better understand the effect of our
  treatment on ropy syrup.
* We should apply the protein expression and purification to a larger production system.
* We should raise the concentration of dextranase in the treatment.
* We should try the enzymatic treatment in larger volume of ropy syrup

References

Kim, Y. M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M. S., . . . Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied microbiology and biotechnology, 91(2), 329-339. doi:10.1007/s00253-011-3201-y

Borkowska, M., Białas, W., Kubiak, M., & Celińska, E. (2019). Rapid micro-assays for amylolytic activities determination: customization and validation of the tests. Applied microbiology and biotechnology, 103(5), 2367-2379. doi:10.1007/s00253-018-09610-0Lagace, L., Camara, M., Leclerc, S., Charron, C., & Sadiki, M. (2018). Chemical and microbial characterization of ropy maple sap and syrup.

Légis Québec. (2012). Règlement des producteurs Acéricoles sur les Normes de qualité et le classement. Légis Québec. Retrieved 11 October 2021, from http://legisquebec.gouv.qc.ca/fr/showdoc/cr/M-35.1,%20r.%2018/20120901#se:17.

Bashari, M., Tounkara, F., Abdelhai, M. H., Lagnika, C., Xu, X., & Jin, Z. (2013). Impact of Dextranase on Sugar Manufacturing and its Kinetic on the Molecular Weights of Remaining Dextran. Sugar Tech, 15(1), 84-93. doi:10.1007/s12355-012-0195-4

Khalikova, E., Susi, P., & Korpela, T. (2005). Microbial dextran-hydrolyzing enzymes: fundamentals and applications. Microbiol Mol Biol Rev, 69(2), 306-325. doi:10.1128/mmbr.69.2.306-325.2005

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# Team:ULaval/Experiments

Project
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Project Description](https://2021.igem.org/Team%3AULaval/Description)
[Experiments](https://2021.igem.org/Team%3AULaval/Experiments)
[Parts](https://2021.igem.org/Team%3AULaval/Parts)
[Results](https://2021.igem.org/Team%3AULaval/Results)
[Safety](https://2021.igem.org/Team%3AULaval/Safety)
[Project Modelling](https://2021.igem.org/Team%3AULaval/Model)
[Engineering Success](https://2021.igem.org/Team%3AULaval/Engineering)
[Proof of Concept](https://2021.igem.org/Team%3AULaval/Proof_Of_Concept)
[Syrup Microbiology](https://2021.igem.org/Team%3AULaval/Syrup_Microbiology)

Human Practices
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Human Practices](https://2021.igem.org/Team%3AULaval/Human_Practices)
[Implementation](https://2021.igem.org/Team%3AULaval/Implementation)

Team
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Team](https://2021.igem.org/Team%3AULaval/Team)
[Attributions](https://2021.igem.org/Team%3AULaval/Attributions)
[Notebook](https://2021.igem.org/Team%3AULaval/Notebook)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

Collaboration & Communication
![](https://static.igem.org/mediawiki/2021/d/de/T--ULaval--Template_iconslide.png)

[Education & Communication](https://2021.igem.org/Team%3AULaval/Communication)
[Collaboration](https://2021.igem.org/Team%3AULaval/Collaborations)
[Contribution](https://2021.igem.org/Team%3AULaval/Contribution)

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Experiments

Medium and reagents

2YT + glucose

LB

Solutions for agarose gels

Preparation Nelson-Somogyi reagent

Cloning

Competent bacteria cells preparation

Competent bacteria cells transformation

Bioshop Taq PCR

Glycerol stock for E. coli

Protein expression and purification

Optimization of protein expression

Verification of protein solubility

Protein expression

Protein purification

Bradford assay

Enzymatic assays

Standard curve of glucose equivalent

Determination of enzymatic unit

Determination of optimal pH

Determination of optimal temperature

Determination of pH stability

Determination of temperature stability

Rheological assays

Rheological assays

Microbiology characterization

Microorganisms isolation

16S & 18S PCR amplification

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# Team:ULaval/Results

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Results

Overview

The experiments we have done during this year can be separated
into four main steps: The first step was the cloning and the confirmation of the E. coli strains for the G. algens
dextranase's expression
([BBa\_K3493000](http://parts.igem.org/Part%3ABBa_K3493000)). Secondly, we have optimized the proteic expression of our dextranase by
testing multiple conditions and thus purified the protein. Thirdly, we realized
micro-SNT enzymatic assays with the previously purified dextranase to characterize its
main properties as pH and temperature for optimal activity. Finally, rheological assays
allowed us to quantify the activity of our dextranase in ropy syrup, an essential test
for the proof of concept for this project. As a part of our contribution to a registry
part, experiments were also performed for the S. mutans
dextranase ([BBa\_K2551003](http://parts.igem.org/Part%3ABBa_K2551003)).

1. Cloning and strains’ confirmation

![](https://static.igem.org/mediawiki/2021/e/e5/T--ULaval--confirm_pET28.png)

Figure 1: Agarose 1% gel with the
diagnostic PCR products for three DH5alpha colonies from the transformation plate.
The transformed plasmid was pET28a-S.mutans. The PCR amplified the full length of
the coding sequence. The expected length was 2900pb. However, we observe that a
colonie expressed a truncated version of the plasmid.

The expression vector pET28a with the cloned sequence of the G. algens and S. mutans
dextranases were ordered from Twist Bioscience. The plasmids were then transformed in
E. coli DH5ɑ competent cells. Clones were used to start
cultures for plasmid purification. One of each purified plasmids was sent to sequencing
to confirm that the sequence on the plasmid was the exact sequence we ordered from Twist
Bioscience. The coding sequence of the G. algens dextranase
corresponded perfectly to
our design, but, surprisingly, the sequencing revealed that the plasmid pET28a-S.mutans
sent had a gap in the coding sequence of our dextranase. To move forward with the
experiments with the S. mutans’s dextranase, we had to
confirm its sequence. So to limit
the expenses related to sequencing the plasmid, we performed PCR on plasmids purified
from three different isolated colonies to identify the ones that contained the full
length coding sequence. The PCR products showed that some of the clones could express a
truncated version of the expression vector. Regarding those results, we selected a
purified plasmid with the full length coding sequence to send to sequencing. The
sequence was then corresponding to our design. Finally, a confirmed plasmid for each
dextranase was transformed in E. coli BL21 competent cells,
which was the strain chosen to perform the expression of the proteins.

![](https://static.igem.org/mediawiki/2021/e/e5/T--ULaval--confirm_pET28.png)

Figure 1: Agarose 1% gel with the
diagnostic PCR products for three DH5alpha colonies from the transformation plate.
The transformed plasmid was pET28a-S.mutans. The PCR amplified the full length of
the coding sequence. The expected length was 2900pb. However, we observe that a
colony expressed a truncated version of the plasmid.

2. Proteins’ expression and purification

For both dextranase, G. algens and
S. mutans, expression’s
optimization experiments were performed to identify the optimal expression condition in
the BL21 strains. The parameters tested were the concentration of the inductor (IPTG),
the induction time and incubation temperature. For each condition and each dextranase,
an aliquot of the induced cultures was collected at different time points: at induction,
after 3h30 of induction and after 18h of induction. Those aliquots were used to estimate
the proteins’ expression by migration of the total extracts on SDS-PAGE gels .

![](https://static.igem.org/mediawiki/2021/f/f2/T--ULaval--confirm_OPT1.png)
![](https://static.igem.org/mediawiki/2021/1/1e/T--ULaval--confirm_OPT2.png)

Figure 2: SDS-PAGE 12% gels with full
extract aliquots from the expression optimization experiment of the G. algens
dextranase. The expected MW was 63.5kDa for the G.algens dextranase. We see the
increase in expression through induction time and the effect of temperature and IPTG
concentration on expression. The induction conditions in which the expression was
the best were 0.

First, the optimal expression’s condition for the dextranase from
G. algens is at a concentration of 0.4 mM or 1mM of IPTG and at an incubation
temperature of 18 °C for 18 hours of induction. The increase of expression over
induction time is revealed by the increase of the protein band corresponding to the
expected weight of the G.algens’ dextranase of 63,5 kDa. We observe that the expression
of the protein is similar in every condition at 37°C, even if there is no inductor in
the medium. This can be explained by the basal rate of expression of the BL21
strain1.
Indeed, the bacteria can express the protein on the expression vector without the
inductor agent but at a low rate. Therefore, we can deduce that 37°C with IPTG is not
optimal conditions for the overexpression because the expression rate is similar to the
basal rate of the strain. The maximal expression observed in our experiment is at an
incubation temperature of 18°C. The lower temperature slowed the growth of E. coli, but
allowed a greater expression of the dextranase because it reduces synthesis rate thus
allowing the protein time to fold properly and avoiding aggregation2.
Moreover, another
way to explain the increase of expression at 18°C is the nature of G. algens which is a
psychrophilic bacteria. The optimal growth temperature for G.
algens is from 10°C to
12°C 3, therefore the induction temperature of 18°C is closer to the native
temperature
in which the dextranase is naturally expressed. In order to determine which condition
between 0.4mM and 1mM of IPTG is the best for induction, we performed a migration of the
soluble fractions and those results will be discussed later.

![](https://static.igem.org/mediawiki/2021/c/c7/T--ULaval--smutans1.png)
![](https://static.igem.org/mediawiki/2021/e/e1/T--ULaval--smutans2.png)

Figure 3: SDS-PAGE 12% gels with full
extract aliquots from the expression optimization experiment of the S. mutans
dextranase. The expected MW was 95.4kDa for the full S.
mutans dextranase. However,
we observe multiple bands related to the induction which indicates proteolysis of
the protein of interest. We see the increase in expression through induction time
and the effect of temperature and IPTG concentration on expression. The induction
conditions in which the expression was the best were 0.4 mM or 1mM of IPTG and at an
incubation temperature of 18 °C for 18 hours of induction.

Furthermore, we were also able to express the dextranase of S.
mutans with the pET28 vector in the BL21 strain. It appears that the optimal expression
conditions for this protein could be at 1mM IPTG, 37°C for 3.5h or at 0.4/1mM IPTG at
18°C for 18h. However, the Comassi staining reaches saturation quickly, so we can not
tell with certainty which condition is the best for expression, because the 3 conditions
described above showed similar staining. For that reason, we have chosen to pursue the
expression’s condition of 0.4mM and 1mM IPTG at 18°C for 18h. As expected, we observe a
protein band at the expected molecular weight for this protein (95,4 kDa), but we also
see a lower weight band appear with the induction which could indicate protein
degradation. The degradation is more important at 18°C. To investigate this result, we
looked into the literature, because this protein had already been expressed and purified
prior to our experiments. In fact, this degradation was also observed by Kim et al.,
2011 4. They also report that the catalytic activity of both fragments is
still active.
Thus, we moved forward with the sonication and the purification for both dextranases.

![](https://static.igem.org/mediawiki/2021/5/53/T--ULaval--algenspurif1.png)
![](https://static.igem.org/mediawiki/2021/7/72/T--ULaval--algenspurif2.png)

Figure 4: SDS-PAGE 12% gels showing
verification of solubility and purification of G. algens
dextranase. A. We compare
the soluble fraction induced with 0.4mM and 1mM IPTG to verify which allows a higher
concentration of the soluble enzyme. We observed that the 1mM condition confears a
larger amount of dextranase in the soluble fraction. B. Gel showing the steps of
IMACS purification. The purified protein seems to be quite pure. However, throughout
the purification we lose a lot of dextranase.

As mentioned above, we used the cell pellets collected from the
optimization experiment (0.4mM & 1mM, 18°C T18) to perform sonication in order to assess
the solubility of the G. algens dextranase for both
conditions. In Figure 4A, we observe
that a larger amount of protein in the soluble fraction is visible for the 1mM condition
than for the 0.4mM condition. The difference between those conditions is the
concentration of IPTG, so we can believe that the higher concentration induced a larger
production of proteins that were not visible with the total extracts due to the Coomassie
staining saturation. With the soluble fraction of the protein expressed at 1mM IPTG 18°C
for 18 hours, we did a first test of purification with IMAC chromatography(Figure 4B).
The SDS-PAGE revealed a high-quality purification of our protein even if there is a loss
of dextranase during elution and washing. After all those verification steps, we
performed the expression experiment of G. algens dextranase
with the optimal condition
(Figure 3) at a larger scale in order to produce an important amount of dextranase. The
purity of this purification was similar to the one shown in Figure 4B, but for a higher
amount of proteins. Then, we quantified the concentration of purified protein with a
Bradford assay. We obtained a concentration of 4.01ug/uL of protein which equals to a
concentration of 636uM of the G.algens dextranase in our 15mL purified sample. We had
enough proteins to start the enzymatic assays. To our knowledge, this is the first time
the dextranase of G. algens is successfully expressed and
purified.

3. Enzymatic assays

Even though both enzymes, our designed dextranase from G. algens
([BBa\_K3493000](http://parts.igem.org/Part%3ABBa_K3493000)) and the one from S. mutans ([BBa\_K2551003](http://parts.igem.org/Part%3ABBa_K2551003)),
were used for the previous
experiments, only the dextranase from G. algens was
characterised by the enzymatic
assays.

Prior to the micro-SNT enzymatic essays, the protocol was verified
using a commercially available dextranase from Penicllium sp. (results not shown). We
were able to confirm the linearity of the assay between 0.008 and 0.072 µmole of glucose
equivalent as previously shown by Borkowska et al. 5. With the protocol
adapted and
functional, we measure the number of enzymatic units of the dextranase from G. algens.
It revealed that for the equivalent of 0.002 µmole of dextranase, on the basis of amount
of protein after purification, has 0.01 U at pH 6.5 and 18 °C. Here, the 1 U is equal to
the release of 1 µmole of glucose equivalent after 1 min. This result show that the
enzyme is active and retains its activity after lysis, purification and storage at 4 °C.
Since this number of enzymatic units added to 0.4 % dextran leaded to the release of
glucose equivalent in the linear region of the assay after 5 min of reaction at 18 °C,
0.01 U was used for the later enzymatic assays.

![](https://static.igem.org/mediawiki/2021/4/47/T--ULaval--ph_enzy.png)

Figure 5:  Activity and stability of the
G. algens’ dextranase in function of the pH at 18 °C
using micro-SNT enzymatic
assays. A. Activity of the dextranase based on the amount of released glucose
equivalent from the dextran T40 under the 0.01 U at pH ranging from 5.5 to 7.0. We
observe that the optimal for the enzyme is at pH 6.5 with 0.036 released glucose
equivalent, but followed closely at pH 6.0 and 7.0 with respective released glucose
equivalent of 0.031 and 0.032. B. Stability of the dextranase under the incubation
at pH between 5.5 and 7.0 for 15 min. The condition where the enzyme seems to be the
more stable is at pH 7.0.

The pH optima were determined using the same method as the
measurement of enzymatic units, but with variations of the pH of the dextran solution.
From the activity’s results measured at pH ranging from 5.5 to 7.0 (Figure 5), the
optimal pH of the G. algens’ dextranase is 6,5. Even though
6.5 is the optimal pH based
on our results, the enzyme is still very active at 6.0 which is promising knowing that
ropy maple is slightly acidic 6. On the other hand, the pH stability was
similarly
obtained with prior incubation of the dextranase at 100 µM in buffer ranging from pH 5.5
to 7 for 15 min at 18 °C. The better condition for the dextranase stability was attained
at pH 7.0, but there is no intense fluctuation in the dextranase’s stability at slightly
acidic pH. In comparison to the amount of released equivalent of glucose, either the
enzymatic activity and stability measured between pH 5.5 and 6.5 are quite stable
between 0.030 and 0.040 µmole. Knowing that the G. algens
dextranase is slightly
impacted in terms of stability at slightly acidic pH where the enzyme has a maximum
activity, we can confirm that the enzyme is appropriate and functional on the basis of
the pH for a treatment in the ropy maple syrup.

![](https://static.igem.org/mediawiki/2021/6/6d/T--ULaval--temp_enzy.png)

Figure 6: Activity and stability of the
G. algens’ dextranase in function of the temperature at
pH 6.5 using micro-SNT
enzymatic assays. A. The activity of the dextranase is based on the amount of released glucose
equivalent from the dextran T40 under the 0.01 U at temperatures ranging from 5 to 50
°C. The enzyme showed to be more active at higher temperatures, reaching 0.104 µmole
of released glucose equivalent at 40 °C and slipping down over 40 °C. At 20 °C, the
activity is lowered to 0.037 µmole. B. Stability of the dextranase under the
incubation at a temperature between 5 and 55 °C for 15 min. We observe that the enzyme
is more stable at lower temperatures, 15 °C and below. The stability seems to
decrease when incubated below 15 °C, but those results appear to be aberrant,
considering that protein stability reaches a plateau at lower temperatures than the
optimal.

Using the same methodology as previously to measure the optimum
activity with temperatures between 5 and 50 °C instead, we were able to determine that
the temperature optimum for the activity of the enzyme is at 40 °C (Figure 6A). Since
the treatment that we propose would be implemented at temperatures between 15 and 20 °C
(see [Implementation](https://2021.igem.org/Team%3AULaval/Implementation)), we can
see that even though the activity is about one-third of the
activity at 40 °C, the enzyme is still very active. This suggests that, while the
enzyme will not degrade the dextran at full capacity, this activity could be enough to
significantly reduce the viscosity of ropy maple syrup to something comparable to maple
syrup on an extended duration of treatment. After 15 min of incubation at each
temperature followed by enzymatic assay at 18 °C for 5 min, we measured that the enzyme
is at its highest stability at 15 °C and lower (considering the aberrant stability at 10
and 5 °C) (Figure 6B). This suggests that keeping the enzyme at 4 °C is enough to
conserve its stability, which is promising for further uses in the industry without
requiring specific and costly conservation conditions. This also shows that a treatment
between 15 and 20 °C would be possible knowing that the enzyme would be close or at its
peak stability but with an activity about one-third of its maximum, which is not
problematic if the treatment is made for an extended period of time.

The enzymatic characterization we have made of the G. algens
dextranase is the first time to our knowledge that the enzymatic properties of this
enzyme were evaluated. Those results are essential for the development of the knowledge
and the use of this dextranase from G. algens. Based on our
results, we are confident
that our dextranase is adapted to the situation of ropy maple syrup which requires
either an enzyme active and stable at pH around 6.0 and 6.5 and an enzyme active and
stable at temperature between 15 and 20 °C.

4. Rheological assays

After characterizing the enzymatic properties of G. algens’
dextranase, we were able to make the proof of concept of the project. Using ropy maple
syrup provided by the PPAQ, the organisation in charge of the market and quality
regulation of maple syrup. We measured the reduction of viscosity of ropy syrup under
the action of our dextranase using rheological assays.

![](https://static.igem.org/mediawiki/2021/2/2a/T--ULaval--rheotot.png)

Figure 7: Viscosity of ropy maple syrup
with and without added buffer and G. algens’ dextranase
using rheological assays.
The assays were performed at 20 °C and 10 °C with prior incubation of the ropy syrup
with the dextranase. The control of the viscosimetric measurement was done with the
same amount of buffer added to the ropy syrup. At 20 °C, the ropy syrup mixed buffer had a viscosity of 0.25 Pa•s while this value was 0.22 Pa•s when mixed with dextranase. The ropy
syrup alone increases in viscosity with increasing fluctuations during the sampling
time. At 10 °C, the ropy syrup mixed buffer had a viscosity of 0.47 Pa•s while this value was 0.42 Pa•s when mixed with dextranase.

To measure the reduction of viscosity, we incubated prior to the
analyses ropy maple syrup with the dextranase at 35.5 µM for 50 minutes at ambient
temperature. In comparison with the measured viscosity of ropy maple syrup with added
buffer (Figure 7), we can see that the sample of ropy syrup under the action of the
dextranase has a decreased viscosity when measured at 20 °C (Figure 6A). This suggests
that not only the enzyme is active in ropy maple as estimated by the enzymatic assays,
but the enzyme is able to significantly reduce its viscosity. To have a better view of
the viscosity reduction after incubation since the relation between viscosity and
temperature is exponential 7, we have done the same analysis at 10 °C with a
prior
incubation this time for 25 min at ambient temperature (Figure 6B). Again, we observed
that treated ropy maple syrup shows a reduced viscosity compared to ropy maple syrup
with buffer. This makes the proof of concept of our project, which is to reduce the
viscosity of ropy syrup for it to be used as a sugar base.

Interestingly, when measuring the viscosity of ropy syrup alone
(Figure 6A), we observed that the viscosity increases during the sampling time with more
important fluctuations. We thought that the high concentration of sugar and dextran
impacted the behaviour of the sample during the analysis. We think that crystallization
of sugar might be responsible for this changing behaviour throughout the time of the
rheological assay, but no further investigation was done since this behaviour was not
observed when adding buffer as a control for the experiment.

Conclusion

In summary, we were able to express and purify our dextranase from G. algens using E.
coli BL21. We used the synthetic gene inserted in a conventional pET28a vector for
protein expression followed by IMAC chromatography. The purified enzyme was
characterized using micro-SNT enzymatic assays that revealed, in the condition of ropy
maple syrup (pH 6.5 and temperature between 15 and 20 °C), that the dextranase would be
adapted for the treatment. It was also confirmed using rheological assays.

References

* Dubendorff, J. W., & Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol, 219(1), 45-59. doi:10.1016/0022-2836(91)90856-2

* Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5(172). doi:10.3389/fmicb.2014.00172

* Bowman, J. P., McCammon, S. A., Brown, J. L., Nichols, P. D., & McMeekin, T. A. (1997). Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic bacteria isolated from antarctic lacustrine and sea ice habitats. Int J Syst Bacteriol, 47(3), 670-677. doi:10.1099/00207713-47-3-670

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