To find a way to organize our project, we decided to follow the Design-Build-Test-Learn cycle (DBTL cycle), a work structure presented to us by 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. Since we want to continue with this project in the 2021 iGEM competition, we focused on the Design phase this year, started the Build phase, and planned the Test and Learn phases. This section shows what we have finished and what we have planned for the next year.
Figure 1: DBTL cycle used to summarize our project idea.
One of our members, Benjamin Ouellet, proposed that we work on a project related to maple syrup production in Canada. Our team was very excited about this idea, so we decided together to work on this topic. We learned that biofilms in the tubes used to collect maple sap can affect the taste of maple syrup, so our first idea was to control or limit the growth of these biofilms. However, our project changed several times along the way. Thanks to the input given by Marie Filteau, Luc Lagacé, Vincent Poisson and Jean-Michel Lavoie, all experts in the maple syrup industry, we decided to change our project idea and work on ropy maple syrup. This syrup is classified as a flavor defect in Quebec’s legislation so it must be destroyed. This syrup also causes environmental and economical losses for maple syrup producers.
We decided to do further research on the problem to gain more insight on how synthetic biology can help address it. Based on our research and discussion with the experts mentioned above, we learned that ropy maple syrup is caused by bacteria that fix to a biofilm found in the tubing system connecting all the maple trees to the sugar shack. These bacteria, when in a fermentative state, start producing dextrans, which are exopolysaccharides that give a ropy and slimy texture to maple syrup when heated during the evaporation process. These bacteria enter the tubing system in different ways: they can come from the tree’s microbiota, the producers when they install new tubing or when leakage occurs in the tubing system and soil microorganisms are introduced by the air flow generated by a vacuum pump.
We also looked at the economic impact of ropy syrup. Since these dextrans can’t be easily detected in the maple sap, maple syrup producers can’t know if or when they will make ropy maple syrup. This means that they only know that their production line is contaminated only after the damage has been done. Because of its thick texture, ropy syrup damages expensive equipment such as the reverse osmosis filter or the boilers (Figure 2). Since ropy maple syrup has no value on the market, producers need to pay for this syrup to be eliminated which causes great economic losses, especially for small producers. We surveyed maple syrup producers in Quebec and confirmed this problem is particularly important for small producers, since in some cases they can lose close to a whole year of profit. Furthermore, given the great amount of energy invested in maple syrup production, the destruction of ropy syrup also means that a lot of energy is wasted. For now the only way producers can lessen the chances of creating ropy syrup is by maintaining good production hygiene, such as washing the tubing system with isopropyl alcohol at the end of the season.
When trying to find a possible solution to this problem, we quickly realised that we could use an enzymatic treatment to remove these dextrans. In nature, many microorganisms, both prokaryotic and eukaryotic, produce dextranases to degrade dextrans found in their environment into many smaller molecules, such as glucose, isomaltose and isomaltotriose (Bourne et al., 1974). Since most of these molecules are either found in or are suspected to have no impact on maple syrup, we agreed that an enzymatic treatment with dextranases would be a good solution. After proposing this solution, we next had to find how to deliver this treatment. We had three candidate places to introduce this treatment, Figure 2 is a diagram summarizing the maple syrup production process:
Figure 2: Diagram showing how maple syrup is produced.
Here, we proposed inserting the dextranase into the tubing system either in the form of a liquid or solid (powder) treatment to eliminate the dextrans as soon as they are produced by the fermentative bacteria. The advantage of this solution is that dextranases might have higher activity at a lower sugar concentration. However, based on our discussions with experts and our survey with producers, we concluded that producers might be reluctant to use such a treatment because it introduces a molecule that modifies all of their maple syrup and they stand by the authenticity and natural aspect of maple syrup. When talking to the MAPAQ, we learned that in Quebec, maple syrup that has been modified in any way can not be sold as maple syrup, but has to be sold under a different name, which means that this syrup would lose a lot of value.
In this scenario, we would introduce our dextranase treatment in the tube found right before the osmosis treatment. This solution could help prevent equipment damage because it degrades the dextrans before they pass through reverse osmosis and the boiler. Here, we think that dextranase would have a higher activity than in maple syrup because of the low sugar concentration. Similar to the scenario above, we think that producers would be reluctant to this solution.
Our idea here is to introduce the dextranase treatment directly into the ropy maple syrup, the final product. This allows producers to keep their maple syrup untouched by this treatment unless it is ropy syrup. The inconvenience here is that the final product will not be considered as maple syrup, although we will be adding value to an otherwise unmarketable product. To sell this product we would have to use terms that exclude “maple” which could be easily done, and is already a common practice. Although this scenario does not help with the damaged equipment, we will still be helping producers by giving them an alternative use for their ropy syrup. Another problem we could encounter here is that ropy syrup isn’t the optimal environment for dextranases. Indeed, ropy syrup is kept at room temperature, has a high viscosity and a high concentration of sugar, which could affect dextranase activity. Our proposed solution would be to perform directed mutagenesis if our treatment isn’t as effective as needed. We decided to go with this solution given that the pros outweigh the cons.
Now that we had our proposed solution, we could work on the details related to this treatment. The first part we worked on was defining the conditions of ropy maple syrup to find what could affect dextranase activity. Here is how we defined the conditions of ropy maple syrup, stored in a typical sugar shack:
- High sugar concentration: Concentration of sugar in maple syrup is measured in Brix degrees (°Bx), where one Brix degree is equal to one percent sugar (mass/mass). Normally maple syrup has a Brix between 65 -67 °Bx, which is a very high concentration of sugar (Ball, 2006). This could affect dextranase activity as sugar can be a potential inhibitor of its activity given that it could be a product of the dextranase’s catalysed reaction. Others have shown that dextranases start losing activity at a sugar concentration of 20 °Bx (Bashari et al., 2013). Discussions with Patrick Lagüe, Stéphane Gagné and Rong Shi, experts in protein structure and function from our university, also confirmed this hypothesis. If we notice inhibition by these sugars, we propose diluting the syrup with water to help with dextranase activity. On the other hand, this would result in a variety of products different by their sugar concentration and it would require additional steps to standardized the final product.
- High viscosity: This is the condition used to define ropy syrup in Quebec, so it’s the characteristic we want to try and remove with our treatment. Based on discussions with the experts mentioned above, there is a chance that viscosity could be a problem for larger proteins because it affects the enzyme’s diffusion. If this is the case, we would try and use a smaller dextranase.
- Low temperature: Most maple syrup producers keep their maple syrup barrels in guarded warehouses at a controlled temperature between 10°C and 15°C. This range of temperature was also confirmed by Luc Lagacé. Given that the most common dextranases are active at a higher temperature, we searched for dextranases that come from psychrophile (cold-adapted) microorganisms.
- Slightly acidic pH: Research has shown that ropy maple syrup has a slightly acidic pH, which is an advantage because dextranases function at a pH slightly below 7 (Khalikova et al., 2005; Lagacé et al., 2018).
With our conditions defined, we were able to start looking for dextranase candidates. As explained above, we needed to find a dextranase that functions at a low temperature. To meet this condition, we aimed to identify dextranases from psychrophilic organisms. Based on structural data from the Protein Data Bank (Berman et al., 2000), we identified two families of dextranases that have very different structures (Larsson et al., 2003; Ren et al., 2019; Suzuki et al., 2012). We submitted the sequences of these proteins to a BLAST search in order to find a list of homologous proteins (Camacho et al., 2009), out of which we selected those that had been taken from psychrophilic organisms (Nogi, 2011). Once we had our candidates, we proceeded to study their sequences and structures through further alignments, homology modelling, and docking analyses to validate the conservation of the active site.(See Model page)
Meanwhile, another part of our team started planning how to overexpress and purify dextranases. We wanted to overexpress the dextranase gene in E. coli because it’s one of the most simple organisms used for protein expression. Thus, we searched for articles expressing dextranase in prokaryotic models. Thanks to this research and discussions with Marie-Ève Picard, a member of Rong Shi’s lab, we decided to use the pET-28a expression vector (Deng et al., 2020; Wang et al., 2016). To summarize, we chose this vector because (1) it’s widely used for protein expression and purification because it adds a Histidine tag to the protein, (2) Dr. Shi’s lab already has the material necessary to work with this vector and will let us use it for our project and (3) purification protocols are widely available for this vector and the dextranase enzyme. As for the purification protocols, we still have a few to sort through to find the best one for our application because we need to find a protocol that is food safe. For now, we will attempt to purify our dextranase with purification protocols that are used in professor Rong Shi’s lab, which concur with some papers discussing dextranase purification (Deng et al., 2020; Wang et al., 2016).
While we wait to gain access to our lab, we plan on ordering expression vectors from Twist Biosciences. For now, we have two dextranases to control: one from Streptococcus mutans and the other from Gelidibacter algens.The dextranase from G. algens was one of the candidates we found during the Design step. G. algens is a psychrophile Gram negative microorganism that can grow at 0°C but has its optimum at between 15°C and 18°C. This organism is also a strictly aerobic chemoheterotroph. The only strain that appears to be studied was found in the Ellis Fjord in the Vestfold Hills in Antarctica (Bowman et al., 1997).
Based on our research, dextranases expressed in vectors have a Histidine tag placed in the C-terminal extremity of the enzyme. To do so, we will have to digest the pET-28a(+) with NcoI and XhoI to remove the Histidine tag and the thrombin site from the N-terminal extremity of the gene. The figure below summarizes the process.
Figure 3: Illustration of the construction of our pET-28a-dextranase plasmid. This construction starts off with the pET-28a vector that is digested with XhoI and NcoI. At the same time, the dextranase gene from G. algens is also digested with XhoI and NcoI to be able to insert it into the linearised pET-28a vector. Note that the size of the dextranase gene here does not correspond to the exact size of the iGEM part we submitted. This is because we had to add restriction sites and a few codons to be able to insert this DNA segment into our vector without affecting codon usage. This process will also be repeated with the dextranase gene from S. mutans. This illustration was created with Snapgene.
As soon as we gain access to the lab and have our vector constructions in hand, we will be able to start testing the activity of our dextranases. First, we will have to test our dextranase expression and purification protocols. To verify its expression levels, we will test our transformed cells on a blue dextran agar plate and if we observe a halo around the colony, it means that it’s able to express dextranase. To verify the purification protocol, we plan on measuring the dextranase activity with a colorimetric test using anthrone, a molecule used to detect reducing sugars in a solution (Koehler, 1954). When we are able to purify enough dextranase, we plan on testing our dextranase activity in different conditions. We plan on testing the effect of temperature, pH and agitation on the dextranase’s stability and activity. Next, we will test the effect of metal ions and detergents on the dextranase’s activity. Last, we will test its substrate specificity by comparing the relative activity of the dextranase when using different substrates. These methods are based on a paper published by Kang et al. (2005). If the dextranases have sufficient activity at a lower temperature, we will be able to test their activity in these different environments: artificial ropy syrup, real ropy syrup and normal maple syrup.The most important feature of our dextranase is its ability to reduce the viscosity of our ropy syrup. As explained in the Design section, viscosity is what makes this type of syrup undesirable, so getting rid of this characteristic will increase the syrup’s quality.
With all the results gathered in the last steps, we will be able to determine if our dextranase candidates have enough activity in ropy maple syrup to transform it into a base without the ropy texture. For now, we’ve come up with the following questions we will need to answer to verify if these dextranases are good candidates to transform ropy syrup.
- Is the expression sufficient to produce enough dextranase?
- Where will the dextranase be found after expression? (organelle, etc)
- Is the final concentration of dextranase after purification enough to follow with the following steps?
- Does the dextranase function in the conditions predicted (temperature, pH, agitation, etc)?
- Is our dextranase capable of removing enough dextran from our artificial and actual ropy maple syrup?
- Are there changes to the purification protocol?
Based on the answers to these questions, we will be able to find what we need to improve with our dextranases.
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