Week 1
We broke up the team to work on three potential projects: Bananas, Biomining, and MICYMouse. We carried out literature review and human practices evaluations to assess feasibility, strengths of the scientific basis of each project and the potential.
MICYMouse:
After doing an in-depth lit review on Team Vilnius-Lithuania’s project from last year, we abandoned our initial idea to build a system using an exolysin. Instead, we found a cool nuclease (NucB) to use instead! We investigated quorum quenching (QQ) as a new method for inhibiting the formation of biofilms - looking into using an AHL acylase to degrade AHL, a quorum signaling molecule. We had an HP meeting with Dr. Gieg to discuss the feasibility of our project. She encouraged us to move forward and gave us more ideas to consider. We are moving forward with a 2-pronged treatment system (QQ + Biofilm degradation) and are currently designing constructs! We’ve scheduled another HP meeting with Dr. Turner (biofilm expert!) for Monday next week! Modelling: There are a few modelling projects that we are considering:
Improving on hydrogenase testing
Molecular docking and molecular dynamics
Measuring and modeling trends in growth rates of biofilm
Biomining:
After investigating metal recovery from electronic waste and having a conversation with Marija, we decided to just focus on recycling REEs from e-waste. We built a biomining pipeline with four major components/subprojects:
Bioleaching: the e-waste will be incubated with acid producing bacteria in a bioreactor that would solubilize the metals. We will be talking to Seb to discuss the details of the bioreactor.
Metal separation: lanmodulin will be immobilized on cellulose beads using a cellulose binding domain. The cellulose beads will then be packed into a column where lanmodulin can separate REEs from rest of the e-waste metal solution
Measurement: We are thinking of using a spectrophotometric assay to measure REE concentration since mass spectrometry is too expensive and inaccessible. We are also looking into designing a measurement system using lanmodulin itself. We scheduled a meeting with Dr. Gailer and Randy to discuss what method of measurement would be most ideal next Monday.
Cantilever system: Identifying specific element based off of oscillation frequency of the cantilever functionalized with lanmodulin
(Tian) Atomic emission spectroscopy (where you burn the metal to plasma and the plasma will have different signatures for each type of metal ion. It could potentially be a cheaper alternative to mass spectrometry). We will have to talk to some analytical chemistry people (meeting next week with Dr. Gailer)
Modelling: There are a few modelling projects that we are considering
Adding a fourth binding pocket to lanmodulin
Increasing the pH at which lanmodulin releases the metal
Increasing affinity of lanmodulin toward a specific lanthanide
Model CBD to determine which protein end is optimal for fusion
Extending the cycle count of lanmodulin
We are currently in the process of figuring out the details/logistics of the above subprojects and we met with Andrew this week to get a better understanding of what we can do.
Bananas:
We first focused on a detection system focusing on HDA amplification (we were going to focus on a HDA detection system, but there’s already a LAMP PCR detection system that works well). However, if HDA is made to be more inexpensive, it could serve to be an alternative to the LAMP PCR. We also focused on finding another wet lab subgroup and have two ideas:
Modifying banana plants (GMO) and be resistant to panama disease
Anti-fungal treatment: Ran into issues with making anti-fungal treatment specific to the desired fungi, because they all have the same biosynthetic pathway
Bio-organic fertilizer: Application of fertilizer can confer resistance to Panama disease. Looking into optimizing organic compounds in the fertilizer to enhance the bacteria’s ability to confer resistance.
Dry lab projects:
DNA amplification and detection kit (hardware): LFA module would be based on iGEM VL design, but not sure about specifics for the DNA amplification module
Deep-learning based detections system for banana diseases (software): Aim is to develop it into a mobile app available on Android OS (as first priority), because that will be more accessible
Week 2
We continued working on the literature review and HP of the Top 3 Projects. We also prepped for the Genome Alberta and yOIL project pitches where each of the projects are pitched and feedback is given on each of the projects.
MICYMouse:
Our week started off with an HP meeting with Dr. Turner (who has expertise in biofilms). This meeting was very helpful and he provided a lot of feedback regarding the feasibility of our project.
Based on his advice, we implemented a third prong to our enzymatic solution: A glucosidase to further target the ECM of biofilms.
Dr. Turner raised some concerns regarding the application of our project (since we hadn't considered multi-species testing/environmental conditions). Thus, we considered some alternative applications ( i.e. water pipelines, medical devices, etc.)
Dr. Turner recommended investigating diabetic foot ulcers, and ways in which we can use engineered bacteria to destroy the biofilm around these wounds to reduce infection and harm. We looked at using bacteria cellulose or hydrogel which contains biofilm reducing enzymes to apply to these wounds.
Biomining:
We met with HP contacts early this week.
Seb, an iGEM alumnus, provided feedback on the whole project, specifically that: (1) the lanmodulin/lanthanide ratio is not a large concern in the context of iGEM competitions, (2) there should be more emphasis on the experiments involving the elution of ions in the column and ways on making that process better, and (3) protein modelling is vital in the reusability of the protein.
He also provided more specific feedback on the bioleaching step, particularly that: (1) the bioreactor design should be rational i.e. start from ideal conditions before adding complexities like reactor parameters and process kinetics
Banana:
In the beginning of the week we finalized the other subproject ideas (bioorganic fertilizer and GMO bananas). We discussed the feasibility of these ideas and what improvements needed to be made for each subproject idea.
We finished the two pitches to Genome Alberta and yOIL and we decided to move forward with the biomining!
Week 3
We split up into three subgroups for the biomining project.
Did a compare/contrast of A. thiooxidans and G. oxydans to determine which is best. In addition, we are waiting for a meeting with Dr. Reed (Thursday @ 10:30 am) to help figure this out further. We might have reached out to another U of T professor (Leslie) who specializes in A. thiooxidans to learn more about the sulfur requirements. We also planned out experimental proof of concept
(decided that bioleaching will be a minor subproject that will be set up in the first few weeks and will then only require weekly up- keep).
For the bioleaching part we are still waiting for the results of the wet lab team on their literature review regarding the microbial species that could be used for the bioleaching step. We identified and contacted potential HP contacts regarding bioprocess and bioreactor design.
Week 4
Based on our HP meeting with Dr. Reed, we decided to try to acquire both G. oxydans and an acidophile so we can compare them and demonstrate that lanmodulin is compatible with multiple bioleaching systems.
Week 1
We broke up the team to work on three potential projects: Bananas, Biomining, and MICYMouse. We carried out literature review and human practices evaluations to assess feasibility, strengths of the scientific basis of each project and the potential.
MICYMouse:
After doing an in-depth lit review on Team Vilnius-Lithuania’s project from last year, we abandoned our initial idea to build a system using an exolysin. Instead, we found a cool nuclease (NucB) to use instead! We investigated quorum quenching (QQ) as a new method for inhibiting the formation of biofilms - looking into using an AHL acylase to degrade AHL, a quorum signaling molecule. We had an HP meeting with Dr. Gieg to discuss the feasibility of our project. She encouraged us to move forward and gave us more ideas to consider. We are moving forward with a 2-pronged treatment system (QQ + Biofilm degradation) and are currently designing constructs! We’ve scheduled another HP meeting with Dr. Turner (biofilm expert!) for Monday next week! Modelling: There are a few modelling projects that we are considering:
Improving on hydrogenase testing
Molecular docking and molecular dynamics
Measuring and modeling trends in growth rates of biofilm
Biomining:
After investigating metal recovery from electronic waste and having a conversation with Marija, we decided to just focus on recycling REEs from e-waste. We built a biomining pipeline with four major components/subprojects:
Bioleaching: the e-waste will be incubated with acid producing bacteria in a bioreactor that would solubilize the metals. We will be talking to Seb to discuss the details of the bioreactor.
Metal separation: lanmodulin will be immobilized on cellulose beads using a cellulose binding domain. The cellulose beads will then be packed into a column where lanmodulin can separate REEs from rest of the e-waste metal solution
Measurement: We are thinking of using a spectrophotometric assay to measure REE concentration since mass spectrometry is too expensive and inaccessible. We are also looking into designing a measurement system using lanmodulin itself. We scheduled a meeting with Dr. Gailer and Randy to discuss what method of measurement would be most ideal next Monday.
Cantilever system: Identifying specific element based off of oscillation frequency of the cantilever functionalized with lanmodulin
(Tian) Atomic emission spectroscopy (where you burn the metal to plasma and the plasma will have different signatures for each type of metal ion. It could potentially be a cheaper alternative to mass spectrometry). We will have to talk to some analytical chemistry people (meeting next week with Dr. Gailer)
Modelling: There are a few modelling projects that we are considering
Adding a fourth binding pocket to lanmodulin
Increasing the pH at which lanmodulin releases the metal
Increasing affinity of lanmodulin toward a specific lanthanide
Model CBD to determine which protein end is optimal for fusion
Extending the cycle count of lanmodulin
We are currently in the process of figuring out the details/logistics of the above subprojects and we met with Andrew this week to get a better understanding of what we can do.
Bananas:
We first focused on a detection system focusing on HDA amplification (we were going to focus on a HDA detection system, but there’s already a LAMP PCR detection system that works well). However, if HDA is made to be more inexpensive, it could serve to be an alternative to the LAMP PCR. We also focused on finding another wet lab subgroup and have two ideas:
Modifying banana plants (GMO) and be resistant to panama disease
Anti-fungal treatment: Ran into issues with making anti-fungal treatment specific to the desired fungi, because they all have the same biosynthetic pathway
Bio-organic fertilizer: Application of fertilizer can confer resistance to Panama disease. Looking into optimizing organic compounds in the fertilizer to enhance the bacteria’s ability to confer resistance.
Dry lab projects:
DNA amplification and detection kit (hardware): LFA module would be based on iGEM VL design, but not sure about specifics for the DNA amplification module
Deep-learning based detections system for banana diseases (software): Aim is to develop it into a mobile app available on Android OS (as first priority), because that will be more accessible
Week 2
We continued working on the literature review and HP of the Top 3 Projects. We also prepped for the Genome Alberta and yOIL project pitches where each of the projects are pitched and feedback is given on each of the projects.
MICYMouse:
Our week started off with an HP meeting with Dr. Turner (who has expertise in biofilms). This meeting was very helpful and he provided a lot of feedback regarding the feasibility of our project.
Based on his advice, we implemented a third prong to our enzymatic solution: A glucosidase to further target the ECM of biofilms.
Dr. Turner raised some concerns regarding the application of our project (since we hadn't considered multi-species testing/environmental conditions). Thus, we considered some alternative applications ( i.e. water pipelines, medical devices, etc.)
Dr. Turner recommended investigating diabetic foot ulcers, and ways in which we can use engineered bacteria to destroy the biofilm around these wounds to reduce infection and harm. We looked at using bacteria cellulose or hydrogel which contains biofilm reducing enzymes to apply to these wounds.
Biomining:
We met with HP contacts early this week.
Seb, an iGEM alumnus, provided feedback on the whole project, specifically that: (1) the lanmodulin/lanthanide ratio is not a large concern in the context of iGEM competitions, (2) there should be more emphasis on the experiments involving the elution of ions in the column and ways on making that process better, and (3) protein modelling is vital in the reusability of the protein.
He also provided more specific feedback on the bioleaching step, particularly that: (1) the bioreactor design should be rational i.e. start from ideal conditions before adding complexities like reactor parameters and process kinetics
Banana:
In the beginning of the week we finalized the other subproject ideas (bioorganic fertilizer and GMO bananas). We discussed the feasibility of these ideas and what improvements needed to be made for each subproject idea.
We finished the two pitches to Genome Alberta and yOIL and we decided to move forward with the biomining!
Week 3
We looked at how to immobilize lanmodulin and talked to Dr. Hu (who works with cellulose) and since we have the support of him, we decided to go with cellulose. The binding domain needs to work in low pH conditions (so we are focusing on that). We also looked at different immobilization methods (that Dr. Park brought up) however, we will focus on cellulose for our project. We also planned out experimental proof of concept.
For modelling we ran all possible mutants through hotspot wizard, found 4 good potential candidates and 4 great, based off of multiple point stability test. The actual software will take more time (we’ll get the candidates done first). We also plan to develop a software which chooses linker position based off of distance and terminus locations (test both sides through binding affinity). We are also looking for lanthanide elemental properties to model lanmodulin-lanthanide binding affinity. We established our homology modelling workflow with chimera for making 3D models of our mutated proteins.
For the separation column we reviewed prior literature on the modelling of fixed-bed adsorbers for the metal separation unit, which will inform the design and physical parameters of the actual physical unit. We contacted Seb for resources he can recommend on bioleaching and adsorption. We also have set up a meeting with him next week for further discussion on the leaching and metal separation units, especially on the workflow from modelling to actual design.
Week 4
This week we still need to find a cellulose binding domain that works in low pH conditions. We also worked on a metal separation plan to determine the experiments that need to be conducted for the next week (separated plan into different phases). We are also working on finding suitable plasmids and creating lanmodulin constructs. We need to also look into experiments on how to determine Kd of the optimized lanmodulin proteins.
We met with Seb early in the week and presented our workflow and from that we determined that we need to conduct experiments for the rational design of the fixed-bed adsorber. We are currently looking into models and experiments we need to conduct for the design of the fixed-bed adsorber: (1) Currently looking into kinetic modelling of the adsorption process first and identify potential experiments to perform in conjunction with the wet lab and (2) will be looking into what fluid mechanics experiments we will be performing and what prototype we should be working with.
Week 1
We broke up the team to work on three potential projects: Bananas, Biomining, and MICYMouse. We carried out literature review and human practices evaluations to assess feasibility, strengths of the scientific basis of each project and the potential.
MICYMouse:
After doing an in-depth lit review on Team Vilnius-Lithuania’s project from last year, we abandoned our initial idea to build a system using an exolysin. Instead, we found a cool nuclease (NucB) to use instead! We investigated quorum quenching (QQ) as a new method for inhibiting the formation of biofilms - looking into using an AHL acylase to degrade AHL, a quorum signaling molecule. We had an HP meeting with Dr. Gieg to discuss the feasibility of our project. She encouraged us to move forward and gave us more ideas to consider. We are moving forward with a 2-pronged treatment system (QQ + Biofilm degradation) and are currently designing constructs! We’ve scheduled another HP meeting with Dr. Turner (biofilm expert!) for Monday next week! Modelling: There are a few modelling projects that we are considering:
Improving on hydrogenase testing
Molecular docking and molecular dynamics
Measuring and modeling trends in growth rates of biofilm
Biomining:
After investigating metal recovery from electronic waste and having a conversation with Marija, we decided to just focus on recycling REEs from e-waste. We built a biomining pipeline with four major components/subprojects:
Bioleaching: the e-waste will be incubated with acid producing bacteria in a bioreactor that would solubilize the metals. We will be talking to Seb to discuss the details of the bioreactor.
Metal separation: lanmodulin will be immobilized on cellulose beads using a cellulose binding domain. The cellulose beads will then be packed into a column where lanmodulin can separate REEs from rest of the e-waste metal solution
Measurement: We are thinking of using a spectrophotometric assay to measure REE concentration since mass spectrometry is too expensive and inaccessible. We are also looking into designing a measurement system using lanmodulin itself. We scheduled a meeting with Dr. Gailer and Randy to discuss what method of measurement would be most ideal next Monday.
Cantilever system: Identifying specific element based off of oscillation frequency of the cantilever functionalized with lanmodulin
(Tian) Atomic emission spectroscopy (where you burn the metal to plasma and the plasma will have different signatures for each type of metal ion. It could potentially be a cheaper alternative to mass spectrometry). We will have to talk to some analytical chemistry people (meeting next week with Dr. Gailer)
Modelling: There are a few modelling projects that we are considering
Adding a fourth binding pocket to lanmodulin
Increasing the pH at which lanmodulin releases the metal
Increasing affinity of lanmodulin toward a specific lanthanide
Model CBD to determine which protein end is optimal for fusion
Extending the cycle count of lanmodulin
We are currently in the process of figuring out the details/logistics of the above subprojects and we met with Andrew this week to get a better understanding of what we can do.
Bananas:
We first focused on a detection system focusing on HDA amplification (we were going to focus on a HDA detection system, but there’s already a LAMP PCR detection system that works well). However, if HDA is made to be more inexpensive, it could serve to be an alternative to the LAMP PCR. We also focused on finding another wet lab subgroup and have two ideas:
Modifying banana plants (GMO) and be resistant to panama disease
Anti-fungal treatment: Ran into issues with making anti-fungal treatment specific to the desired fungi, because they all have the same biosynthetic pathway
Bio-organic fertilizer: Application of fertilizer can confer resistance to Panama disease. Looking into optimizing organic compounds in the fertilizer to enhance the bacteria’s ability to confer resistance.
Dry lab projects:
DNA amplification and detection kit (hardware): LFA module would be based on iGEM VL design, but not sure about specifics for the DNA amplification module
Deep-learning based detections system for banana diseases (software): Aim is to develop it into a mobile app available on Android OS (as first priority), because that will be more accessible
Week 2
We continued working on the literature review and HP of the Top 3 Projects. We also prepped for the Genome Alberta and yOIL project pitches where each of the projects are pitched and feedback is given on each of the projects.
MICYMouse:
Our week started off with an HP meeting with Dr. Turner (who has expertise in biofilms). This meeting was very helpful and he provided a lot of feedback regarding the feasibility of our project.
Based on his advice, we implemented a third prong to our enzymatic solution: A glucosidase to further target the ECM of biofilms.
Dr. Turner raised some concerns regarding the application of our project (since we hadn't considered multi-species testing/environmental conditions). Thus, we considered some alternative applications ( i.e. water pipelines, medical devices, etc.)
Dr. Turner recommended investigating diabetic foot ulcers, and ways in which we can use engineered bacteria to destroy the biofilm around these wounds to reduce infection and harm. We looked at using bacteria cellulose or hydrogel which contains biofilm reducing enzymes to apply to these wounds.
Biomining:
We met with HP contacts early this week.
Seb, an iGEM alumnus, provided feedback on the whole project, specifically that: (1) the lanmodulin/lanthanide ratio is not a large concern in the context of iGEM competitions, (2) there should be more emphasis on the experiments involving the elution of ions in the column and ways on making that process better, and (3) protein modelling is vital in the reusability of the protein.
He also provided more specific feedback on the bioleaching step, particularly that: (1) the bioreactor design should be rational i.e. start from ideal conditions before adding complexities like reactor parameters and process kinetics
Banana:
In the beginning of the week we finalized the other subproject ideas (bioorganic fertilizer and GMO bananas). We discussed the feasibility of these ideas and what improvements needed to be made for each subproject idea.
We finished the two pitches to Genome Alberta and yOIL and we decided to move forward with the biomining!
Week 3
A conversation with Robert revealed that we will have access to a plasma mass spectrometer and a luminometer. We decided to go with the nanobit reporter system (from the meeting with Robert). We still have to look into different reporter systems (electrochemical system) in case the nanobit system doesn’t work. We also planned out experimental proof of concept.
We also have to develop a smaller linker for the modelling team to determine what is the best linker we should use.
For the biosensor hardware we are researching past iGEM biosensor designs, looking for the most appropriate design for the project.
Week 4
We are looking more into the electrochemical signal for the reporter system (still need lots of work for this). Currently, we have a preliminary idea for the experiments that will need to be carried out (will be very similar to the metal separation subgroup) and we are also designing constructs for the nanobit reporter system.
For the biosensor, the circuit is almost done, now looking into building a chassis.
Week 1
We broke up the team to work on three potential projects: Bananas, Biomining, and MICYMouse. We carried out literature review and human practices evaluations to assess feasibility, strengths of the scientific basis of each project and the potential.
MICYMouse:
After doing an in-depth lit review on Team Vilnius-Lithuania’s project from last year, we abandoned our initial idea to build a system using an exolysin. Instead, we found a cool nuclease (NucB) to use instead! We investigated quorum quenching (QQ) as a new method for inhibiting the formation of biofilms - looking into using an AHL acylase to degrade AHL, a quorum signaling molecule. We had an HP meeting with Dr. Gieg to discuss the feasibility of our project. She encouraged us to move forward and gave us more ideas to consider. We are moving forward with a 2-pronged treatment system (QQ + Biofilm degradation) and are currently designing constructs! We’ve scheduled another HP meeting with Dr. Turner (biofilm expert!) for Monday next week! Modelling: There are a few modelling projects that we are considering:
Improving on hydrogenase testing
Molecular docking and molecular dynamics
Measuring and modeling trends in growth rates of biofilm
Biomining:
After investigating metal recovery from electronic waste and having a conversation with Marija, we decided to just focus on recycling REEs from e-waste. We built a biomining pipeline with four major components/subprojects:
Bioleaching: the e-waste will be incubated with acid producing bacteria in a bioreactor that would solubilize the metals. We will be talking to Seb to discuss the details of the bioreactor.
Metal separation: lanmodulin will be immobilized on cellulose beads using a cellulose binding domain. The cellulose beads will then be packed into a column where lanmodulin can separate REEs from rest of the e-waste metal solution
Measurement: We are thinking of using a spectrophotometric assay to measure REE concentration since mass spectrometry is too expensive and inaccessible. We are also looking into designing a measurement system using lanmodulin itself. We scheduled a meeting with Dr. Gailer and Randy to discuss what method of measurement would be most ideal next Monday.
Cantilever system: Identifying specific element based off of oscillation frequency of the cantilever functionalized with lanmodulin
(Tian) Atomic emission spectroscopy (where you burn the metal to plasma and the plasma will have different signatures for each type of metal ion. It could potentially be a cheaper alternative to mass spectrometry). We will have to talk to some analytical chemistry people (meeting next week with Dr. Gailer)
Modelling: There are a few modelling projects that we are considering
Adding a fourth binding pocket to lanmodulin
Increasing the pH at which lanmodulin releases the metal
Increasing affinity of lanmodulin toward a specific lanthanide
Model CBD to determine which protein end is optimal for fusion
Extending the cycle count of lanmodulin
We are currently in the process of figuring out the details/logistics of the above subprojects and we met with Andrew this week to get a better understanding of what we can do.
Bananas:
We first focused on a detection system focusing on HDA amplification (we were going to focus on a HDA detection system, but there’s already a LAMP PCR detection system that works well). However, if HDA is made to be more inexpensive, it could serve to be an alternative to the LAMP PCR. We also focused on finding another wet lab subgroup and have two ideas:
Modifying banana plants (GMO) and be resistant to panama disease
Anti-fungal treatment: Ran into issues with making anti-fungal treatment specific to the desired fungi, because they all have the same biosynthetic pathway
Bio-organic fertilizer: Application of fertilizer can confer resistance to Panama disease. Looking into optimizing organic compounds in the fertilizer to enhance the bacteria’s ability to confer resistance.
Dry lab projects:
DNA amplification and detection kit (hardware): LFA module would be based on iGEM VL design, but not sure about specifics for the DNA amplification module
Deep-learning based detections system for banana diseases (software): Aim is to develop it into a mobile app available on Android OS (as first priority), because that will be more accessible
Week 2
We continued working on the literature review and HP of the Top 3 Projects. We also prepped for the Genome Alberta and yOIL project pitches where each of the projects are pitched and feedback is given on each of the projects.
MICYMouse:
Our week started off with an HP meeting with Dr. Turner (who has expertise in biofilms). This meeting was very helpful and he provided a lot of feedback regarding the feasibility of our project.
Based on his advice, we implemented a third prong to our enzymatic solution: A glucosidase to further target the ECM of biofilms.
Dr. Turner raised some concerns regarding the application of our project (since we hadn't considered multi-species testing/environmental conditions). Thus, we considered some alternative applications ( i.e. water pipelines, medical devices, etc.)
Dr. Turner recommended investigating diabetic foot ulcers, and ways in which we can use engineered bacteria to destroy the biofilm around these wounds to reduce infection and harm. We looked at using bacteria cellulose or hydrogel which contains biofilm reducing enzymes to apply to these wounds.
Biomining:
We met with HP contacts early this week.
Seb, an iGEM alumnus, provided feedback on the whole project, specifically that: (1) the lanmodulin/lanthanide ratio is not a large concern in the context of iGEM competitions, (2) there should be more emphasis on the experiments involving the elution of ions in the column and ways on making that process better, and (3) protein modelling is vital in the reusability of the protein.
He also provided more specific feedback on the bioleaching step, particularly that: (1) the bioreactor design should be rational i.e. start from ideal conditions before adding complexities like reactor parameters and process kinetics
Banana:
In the beginning of the week we finalized the other subproject ideas (bioorganic fertilizer and GMO bananas). We discussed the feasibility of these ideas and what improvements needed to be made for each subproject idea.
We finished the two pitches to Genome Alberta and yOIL and we decided to move forward with the biomining!
Week 3
No further updates for this week.
Week 4
For the bioleaching aspect we met with Dr. Reed and determined some considerations on the bioreactor design. We will be meeting with Seb again to discuss more about the bioreactor design (i.e. if it will follow the workflow for the fixed-bed adsorber).
Week 1
During this week, we worked to complete our experimental plan and figured out what reagents/procedures to use. We had Tian and Tammy check it over this list. Next week we Will be meeting with an HP contact (Caleb Stetson at 1pm on Monday- analytical chemistry postdoc) to finalize some of the chemical details of our analytical techniques. Currently finishing up the order list for Monday.
Week 2
For bioleaching we worked to refine and finalize experimental plans! We have fun plans in place to dissolve our metal in funky acid and work on characterizing the magnets and the practicing arsenazo assay, and make media to culture our cells in when they arrive. For bioleaching, we briefly looked into potential workflows for developing bioleaching unit prototypes. Right now we are waiting on experimental results to see what species we will be choosing for bioleaching.
Week 3
Culture media for both organisms are being prepared today and will be autoclaved over the weekend. We will also be starting the demagnetization process on the magnets soon.
Week 4
No further updates for this week.
Week 1
This week we worked to complete our experimental plan and figured out what reagents/procedures to use! We also finished designing the constructs and had Tian, Tammy, and Sravya check over these! Currently finishing up the order list for Monday and we are also meeting with Joey on Monday at 2 PM.
For protein modelling, we are working on getting kinetic rate constants from an ML model. Also currently working on a linker position finder in order to maximize the distance between the linker and binding pockets to reduce damage on binding affinity.
For the separation column modelling, we are starting on a base case for the fixed-bed adsorber by initially assuming process parameters and material properties from similar systems. We will also perform a sensitivity analysis for the models governing the fixed-bed adsorber design to determine which parameters dramatically affect the output parameters.
Week 2
We had a meeting with Joey. We also looked at potential cellulose binding domains (CBD) and the issue of cellulose metal adsorption (might look at other immobilization methods as well). We worked on the order list and determined the reagents we need for our metal separation plan!
This week we talked with Seb and finalized a good starting point for both modelling and experimentation. The baseline adsorption of REEs by raw cellulose beads (cellulose beads without lanM) will be significant, so appropriate measurements must be undertaken for it to be considered in the design. We are also looking into a prototype metal separation unit; however, we are still looking at potential materials meeting certain criteria (e.g. vessel transparency, material cost, etc.) for performance evaluation compatibility. Right now we are digging into the mathematics behind the adsorption column modelling (the current model only considers monovalent adsorption, so we have to account for lanM’s trivalent binding capacity). Also, currently looking into a machine learning scheme to predict the kon and koff rate constants as they are needed for the adsorption unit modeling.
Week 3
We were planning on using a cellulose binding module (CBM) family II for immobilization of LanM but we had a meeting with Dr. Hu this week in which he suggested that we should use a family III CBM as it would be easier to express in E. coli.
We started writing the code for the mathematical simulation of the fixed-bed adsorber. We are currently learning about how to implement the numerical method (orthogonal collocation on moving finite elements) required to solve the system of partial differential equations governing the adsorber unit. We talked with Dr. Hu to get more details on the experiments we can perform on the unit and on the cellulose beads. He said that the adsorber column is too complex a system for us to run experiments on. He suggested starting experiments on simpler systems i.e. raw cellulose beads and functionalized cellulose beads and waiting for the results before performing the fixed-bed adsorber experiments.
Week 4
For modelling, we developed an ML model that predicts the rate constants of a given protein so that we can get the Kon and Koff of lanmodulin. The Kon and Koff values are needed for the modeling of the absorption of lanthanides in our reactor column. We are working on reducing the number of features required per each protein to make a prediction.
For drylab, we used FEniCS to numerically approximate the solution to the coupled PDEs for the adsorber unit. We aim to get the functions for the breakthrough curve, which will yield the concentration of the effluent as a function of time. We are still learning ParaView to visualize the results of the finite element solution. We initiated writing out the protocols to characterize baseline binding of REE onto raw cellulose beads i.e. non-functionalized cellulose beads in a flask and as part of the adsorber column
Week 1
This week we worked to complete our experimental plan and figured out what reagents/procedures to use! We also finished constructs and will have Tian and Tammy take a look.
For protein modelling, the homology modelling has been a little problematic, and we are hopefully developing accurate models this afternoon, then doing binding affinity evaluation after.
For the biosensor hardware, parts were ordered for testing. We will use ESP32 instead of Arduino Uno (same IDE but better MCU in general). Also, the two sensors will be tested: TSL237 and IR-blocking photodiode.
Week 2
During the week we contacted multiple professors from the chemistry department at the University of Calgary in order to validate our electrochemical system for measuring the lanthanide concentration. We also updated the regents list with everything we need for our experiments. The hard deadlines for constructs is the end of Monday, June 14 for the measurement subgroup.
For the biosensor, the orders came in and tests started yesterday. The analog sensor is kind of hard to use, so there might be an issue with wiring. We are going to test with an op-amp today to see if it is a sensitivity issue.
Week 3
During the week, we had two important meetings regarding the electrochemical system. One with Dr. Sutherland who we presented our electrochemical signal to and a iGEM team in Brazil who is also utilizing an electrochemical signal for use in their biosensor. Currently for the electrochemical system, protocols are still being researched upon in regards to what signal molecule will be used for the oxidation-reduction reaction that needs to take place in order to create the standard curve we need. Issues that we are running into is finding ways to selectively bind the signal molecule to the terminal end of the lanmodulin protein. For the nanobit construct, modelling helped with choosing linkers for us and we have finished the construct for that. We need to order that so we can work on it as soon as possible. For the BRET system we are currently still looking into what linkers to use for that.
For protein modelling, we produced 125 homology models for 25 sequences (5 different linkers). We filtered them through visual inspection, produced worst case and best case models for each sequence. We also wrote a script to measure all distances and to produce a variety of sequences and did a binding affinity evaluation for the remaining. We then found a great construct and sent it to the wet lab team. We also got access to arc, verifying distance range for chosen construct through MD. We’re still working on the primary structure for lanM.
For the biosensor, we are now using transimpedance amplifier design to provide more stability (original design was pretty similar but less stable). We are still in the process of tweaking some parts, doing simulations, and calibrating to find the best set of values.
Week 4
During this week we worked on developing the protocols for the BRET/Nanobit system. In addition, we picked two promising protocols to develop the electrochemical measurement system (immobilizing the lanmoulin on the gold electrode and attaching the ruthenium signal molecule on the other end of lanmodulin), and will send those over to Dr. Sutherland so he can take a look over the protocols for us and give us feedback. We ordered the nanobit construct and are waiting on the modelling team for the BRET system linkers.
For modelling, we ran MDs for the primary structure of LanM as well as for the lanmodulin-luciferase fusion protein. For choosing a linker for the BRET system, its size is concerning because of how small it makes lanmodulin. Our workflow doesn't work on it. Running three different types of homology R-fold, comparative modelling, ab initio. They're taking a long time but good results so far.
For the biosensor, the circuit on Arduino is almost done, just need to add an LCD screen for UI. We are having issues moving the system from Uno to ESP32 - the previously ordered board is faulty. Various fixes suggested online have been tried but none of them helped. A new ESP32 board on the way, but I also messaged the company that sold the faulty board. In the worst case scenario - we just attach a Bluetooth module to the Arduino Uno (less than ideal, but good enough for our plan).
Week 1
For bioleaching, we decided that we will wait for the preliminary results of the A. thiooxidans and G. oxydans wet lab experiments before deciding to model the bioreactor. We will also be reading more about general bioreactor design workflow while waiting for the wet lab results.
Week 2
No further updates for this week.
Week 3
We started looking into open-source designs for the bioreactor unit. We plan on developing a workflow that will allow us to integrate important bioleaching-specific parameters into modifying existing open-source bioreactor designs.
Week 4
We decided that the bioreactor design will be general, i.e. capable of supporting growth of transformed E. coli and bioleaching microbes. Operating parameters can just be varied to support a specific species, but the general reactor will stay the same. We started looking at measurement protocols to give to the wet lab, so we can extract values necessary for characterizing the growth curves of the microbes. For both protein production and bioleaching processes, the growth conditions will be mimicked in the bioreactor.
Week 1
We have been working hard on developing and improving the protocol for the arsenazo assay (an assay to determine neodymium concentration in various media and buffers). We are doing the full set of 120 samples with all 5 medias/buffers, time permitting. Our cells still have not arrived yet.
Week 2
We are still waiting for bugs to arrive and on ICP-MS details to begin acid digestion. We finished arsenazo stuff and the calibration curves look good!
Week 3
No further updates for this week.
Week 4
No further updates for this week.
Week 1
The Birnam Wood (Random Forest Model) was an ML program used for predicting Kon and Koff Values. We developed a cross nested validation algorithm (which brings down ML iterations from 201! to 2012). The goal is to have it finished by the end of next week.
We finally figured out how to work with FEniCS through WSL; FEniCS tutorial is done. Right now in the process of writing out the protocol for the adsorption experiments on the cellulose beads. We plan on using individual ion solutions of Nd and Fe as the test solutions to determine the adsorption capacity of cellulose beads for these metals (this is the alternative since we do not have access to ICP-MS, which will allow the determination of concentrations in a complex metal solution). We will be using the Arsenazo assay for determining the concentration of Nd; still looking at Phenanthroline assay to measure concentration of Fe. We have to make sure the e-learning is up to date so that bioreactor people can go into the lab and carry out wet lab experiments.
Week 2
We have been troubleshooting protein production in the lab. We will be running a positive control with 6GIX (2019 iGEM protein) next week to verify our protocol. We have also been troubleshooting our cPCRs. We got it to work twice in a row with new primers but it stopped working.
For modelling, the Birnam Wood is done for the most part. We are still working on the parameters to get accurate Kon and Koff values for LanM and neodymium interaction. Looking for more projects - ideation happening now.
We did the implementation of ODE equations into python and generated two plots for rate of solute adsorption vs. solute concentration on adsorbent graph, and concentration of solute on adsorbent vs. concentration of solute in the liquid in the pores graph. Still have two more PDEs to solve.
Week 3
We worked on producing 6Gix and Deirdre’s (lab technician) GFP. Ran it on the SDS gel and GFP worked while 6Gix didn’t. Thinking about putting LanM into a different plasmid since GFP was working, that means our induction methods probably work. We planned experiments for next week and have new troubleshooting methods. We will experiment with a new method of lysing the cells and can hopefully produce the protein!
For protein modelling, we are writing a paper for the iGEM Maastricht collaboration on the kinetic rate constant prediction software. We are one program away from getting all the parameters. For the cellulose binding module 6Mi5 complex, we are making sure binding pockets are still strong.
We met with an HP contact for mechanistic modelling for the adsorption column, and they said that it will be trickier than anticipated. We will work on this in the background for now. Right now we are finding literature values for parameters and decided that analytical models might be the way to go for now. Although this will not be able to reveal much about the mechanism, we can easily relate this to experimental data.
Week 4
No further updates for this week.
Week 1
The Nanobit construct came so it has been successfully transformed into BL21 and DH5a, a miniprep and PCR have also been done with the construct. We will start protein purification soon and prepping protocols to test the first measurement system. Currently still working on the electrochemical system. In contact with Dr. Sutherland on developing the protocols for developing that measurement system. Right now, looking into how to characterize that the ruthenium nanoparticle has been attached onto the end of lanmodulin. Also looking into the LacZ electrochemical system as an alternative.
For protein modelling, we choose optimal linkers for the BRET system using ideal measurements. MD for verification is done, and we are going to measure the distances every 10ps for more accuracy.
For the biosensor, the whole system now runs on ESP32. There are still some minor issues that need fixing and a few more additions to hardware, but they should be easy to figure out. Now programming an Android app to receive data from the sensor via Bluetooth. The next steps is chassis design and 3D printing (Cedric has friends that have 3D printers and is part of a club). We need to figure out how to convert frequency output into units of light intensity.
Week 2
We are currently holding off on ordering the BRET system as in-lab protein purification is having issues. We have the successful transformation of the NanoLuc system. We finished researching everything for the electrochemical system, we need to run over one last experimental design with Tian and Dr. Sutherland before ordering all needed reagents.
For the biosensor, we are working on Bluetooth connection with an Android phone. We also need a measurement and calibration page on the phone app (Jonathan has volunteered to help out with this).
Week 3
We are currently looking into purifying the NanoBIT protein. We had a successful transformation and sequencing read, indicating that our desired construct was transformed into bacteria. We’re currently troubleshooting protein purification (see metal separation) before we want to order our BRET construct.
For the biosensor, the BLE connection is up and running! We are now developing UI for app. We still need to work on the calibration algorithm, and waiting on the measurement team for details on units. We are designing the sensor body soon, Seb offered to print it but we can also look into Makerspace since we plan to print a lot of parts. A few more additions to hardware are needed, but we are almost ready to solder the parts and have a final product. We also have to look into entrepreneurship (can make the biosensor for like a water treatment centre).
Week 4
No further updates for this week.
Week 1
We wrote out the protocol for measuring the growth rate of E. coli (untransformed, transformed but not induced, and transformed and induced) with the help of Seb. We will be talking to Tian later today about the protocol and the use of the plate reader.
Week 2
We did the first attempt at replicating the protein production model found in paper. We need to do inventory and make a shopping list for the basic bioreactor model hardware components. We also now have an IDB card that we can use to purchase supplies from engineering stores.
Week 3
A two-stage model (non-induced and induced stage) for the growth rate of transformed E. coli in a bioreactor operated in batch mode has been implemented. We are setting up the workflow for the design and construction of the bioreactor prototype. We are also working with Cedric regarding the automation of recording measurements of temperature, aeration rate, pH, etc.
Week 4
No further updates for this week.
Week 1
No further updates for this week.
Week 2
We have ICP-OES access now, so that’s exciting. MTAs for the bacteria are almost ready; University of Calgary legal has the paperwork.
Week 3
No further updates for this week.
Week 4
We received G. oxydans last week, and we are starting on experiments with it now!
Week 1
We emailed Dr. Hu about cellulose materials but he still has not yet responded. The G-block of lanmodulin will be ordered so that it can be directly cloned into a vector for protein production troubleshooting.
For software we are writing up the Birnam Wood paper for the iGEM Maastricht journal. We are working on new software which predicts the number of functionally or partially functioning binding pockets in protein (initially hardcoded by detecting D pads, once sufficient data is gathered, we will use machine learning to make predictions).
For the preliminary implementation of the Bohart–Adams model and Thomas model, we are currently replicating results from the paper they were derived from. The next step is to perform a bit more literature review to see (a) which one is more accurate for liquid adsorption and (b) which model has more easily measurable parameters.
Week 2
We are still troubleshooting protein production. We are trying to clone in our constructs into Tony’s vectors. We did get a few colonies that passed the initial screening. We are in the process of screening the colonies using PCR. So far we haven't had a positive colony.
For software, we worked on the EF hand time series classifier and predictor. We are planning on using a long short-term memory recurrent neural regressor. We worked on gathering sufficient data, then formatting it into the correct format. Hopefully we can get it running by the end of next week.
For the adsorber column, we are finishing the sensitivity analysis.
Week 3
No further updates for this week.
Week 4
We are still working on/troubleshooting protein production in the lab.
For software, we are adding to the database for the EF-hand finding program and working on the ML model.
The preliminary adsorber column modelling for the dimensions of the prototype is done (performed this while writing the Maastricht journal). The way it was done for the paper was very rudimentary - we will probably use Gradient Descent to optimize for the length and diameter to minimize the pressure-to-flow rate ratio. The next step is to meet with Andrew for quantifying the sensitivity parameters.
Week 1
We are meeting with Robert in regards to the electrochemical system early next week. We are currently working on the flow of experiments for all the measurement systems.
For the biosensor, we tried adding buttons but it burned the microcontroller. We are still trying to figure out why, but we might end up going for a different approach. We finished the CAD model for the sensor, and will print soon and currently working on Maastricht journal this weekend.
Week 2
We met with Robert from Fredsense and have a good idea of what to order for the electrochemical system. Finished updating the measurement subgroup journal with protocols that are needed in order to make it. Currently looking at what gold electrodes to order.
For the biosensor, we finished the draft for the Maastricht journal. We are still figuring out if a toggle switch is better than a button for UI. We talked to the measurement team about how calibration will work.
Week 3
No further updates for this week.
Week 4
We are still working on/troubleshooting protein production in the lab.
For the biosensor, we are having issues with one print in regards to printing the chassis. Currently we are making the calibration algorithm. One more hardware component (slide switch) will be added to the sensor. In addition, we realized that the UI might be a little hard with the program we’re using to make the app.
Week 1
We implemented the biomass growth model for a substrate-limited culture but moved away from this because it is too simple. We implemented the three-block (biomass growth, IPTG uptake, and protein production) model for E. coli cultivation in a fed-batch bioreactor. Terms accounting for change in volume complicated the model, which we found unnecessary to include in ours since we are only operating it in batch mode. Currently rewriting the equations for batch mode only. The next step is to perform sensitivity analysis on the literature parameters (to see if they affect predictions significantly) and experimental variables (to see if performing experiments is needed).
Week 2
No further updates for this week.
Week 3
No further updates for this week.
Week 4
We are ordering the materials today!Right now we have a deterministic model of the bioreactor. We have to make the bioreactor model stochastic for the parameters we find in literature or cannot reliably measure in the lab.
Week 1
No further updates for this week.
Week 2
No further updates for this week.
Week 3
We set up controlled aqua regia experiments. Additionally, we set up two treatments of bioleaching with G. oxydans.
Week 4
We are running experiments on acid generation and metal dissolution in G. oxydans. We are also planning to finally use the ICP-OES this week to verify the Arsenazo assay and validate our initial bioleaching experiment results.
Week 1
No further updates for this week.
Week 2
We have been cloning our LanM gblock into the expression vectors. This should give us a new fusion protein with a solubility tag (GST) that might help LanM be expressed better.
For the EF hand-finder, we created a convolutional neural network with a long short term memory network. Currently working on being able to submit a whole protein sequence and being able to output the number of efhands present in the sequence (currently our model has 97% accuracy).
For the adsorber column, the first run at sensitivity analysis was a success (thanks to Andrew!). Things to note is that the sensitivity indices change as a function of time, the kinetic constant is important at the very beginning and at the very end, and the sorptive capacity is important during actual breakthrough. We have to modify the sampling method: We tried out applying this to a system of ODEs - results were weird looking but we will look more into that. The next steps are to reach out to a PDE professor about a numerical approach to getting the breakthrough behavior of the column, figure out what’s wrong in the code when using SALib on ODE systems, and go into the lab this week to finish cellulose adsorption experiment.
Week 3
We have positive GST-LanM colonies (with the sequence verified)! We will be checking for protein production tomorrow.
For software/protein modelling, we made a breakthrough in our EF prediction system (we are now able to feed in full protein sequences and receive a prediction on the number of EF hands and the specific location of them). It is not as accurate as we would like it to be, so further tuning is required. We are also looking for another potential software or modelling project.
For the batch adsorption experiment, we performed two tests one was a mass-transfer test and the second one was a preliminary desorption test. One thing to note is that forced convection promotes adsorption of Nd3+ onto cellulose and e are inverting the tube to suspend cellulose in the solution as it is enough for it to reach equilibrium. We are using a low pH solution that desorbs Nd3+ from cellulose by up to 50%. We will have to think about a way to talk about how LanM has a competitive edge to cellulose despite cellulose adsorption of lanthanides being pretty good. For the breakthrough curve we will be using COMSOL instead of FEniCS.
Week 4
We are going to do a western blot on Monday to determine whether we have protein (SDS-PAGE gel looks promising so far). We are planning on running protein characterization assays on these as well.
For software, we are working on a genetic algorithm for creating the ideal 4th binding pocket motif. We’ll be using the EF hand software that we created to evaluate the pocket.
For the adsorber column, we got access to COMSOL this week! We simulated how the beads get saturated with lanthanide ions with a cool animation. One thing to note is that the direction of bead saturation depends on the magnitude of the flow velocity relative to how fast the binding reaction takes place.
Week 1
No further updates for this week.
Week 2
We are still waiting on successful protein production. We have not ordered BRET or the electrochemical system because we’re not sure if we should, but we’re going to start prepping the G-blocks so we can order it as soon as we’re ready.
For software/protein modelling, we are testing/validating EF hand finder with the measurement group proteins.
For the biosensor, we received most of the external components for the sensor and we’re ready for sanding. There are two more external parts plus all the internal parts that need to be printed. We also just added a slide switch to the circuit.
Week 3
We made a draft of the BRET system gblock. We need to determine how much free DNA do we have/get because we would like to order multiple g-blocks (3 gblocks) for the electrochemical system, but have to determine if that is possible). Also, we are currently working on wet lab troubleshooting.
For the biosensor, we added a slide switch for operation mode and the the code to make it work is on the Arduino IDE. We are still waiting on a few more parts to be printed; thinking of contacting Makerspace again.
Week 4
The G-blocks for our constructs (both BRET and electrochemical) were ordered - we’ll begin cloning once they come in.
For the biosensor, we are still waiting for the 3D models to be printed
Week 1
No further updates for this week.
Week 2
We are still waiting for the materials to come in.
Week 3
For the hardware aspect, all materials have arrived except for the air filter! We will be using a makeshift air filter (out of syringe and cotton) for now.
Week 4
Currently working on automating sample collection with peristaltic pump and arduino controller. We had to order an extra motor driver so it is a work in process currently.
Week 1
Planning to use the ICP-OES on Monday (samples have been prepared during this week).
Week 2
No further updates for this week.
Week 1
In the lab there was LanM successful production! Constructs with the cellulose-domains have been ordered.
For the software, we are working on three different algorithms for mutating lanmodulin’s binding pockets.
For the adsorber column: The graph for lanM-Nd3+ binding kinetics are done. The initial breakthrough curve has been determined. Also finished performing parametric sweeps! We are currently trying to visualize how the breakthrough curve changes as a function of different parameters by plotting the data points.
Week 2
In the lab we ran first series of metal separation, but we still need more data and to optimize the experiment.
We produced over a 100 million potential EF hands for our fourth binding pocket! We only evaluated about 3 million though, but we just need to adjust some things to get the full results and develop big diagrams. Because of that fourth binding pocket wiki writing may take a little longer. But we just finished birnam wood if someone can edit it and are working on EFHE.
For the adsorber column model, we had a dummy value last week for the kinetic rate constants, and it worked out great! Now we are using the kinetic rate constants from Birnam Wood, but we are not getting the curves we want. Currently tinkering with the flow velocity through the column so Nd3+ actually has time to: (1) diffuse through the beads, (2) bind to lanM (we think this isn’t the problem since the binding is super fast). We also finished cellulose adsorption experiments last week! We determined that water alone does not desorb Nd3+ from cellulose. This means that lowering the pH of the washing solution causes the desorption, not just presence of the fluid itself!
Week 1
Measurement G-block constructs should be in (delivered) sometime next week, we’ll be starting the wet lab workflow with it ASAP.
For the biosensor, we are working on a function to convert frequency into concentration (still in progress).
Week 2
The measurement g-blocks came in (NanoBiT, BRET, and electrochemical system), we just have to transform the bacteria with our ligations next week and start protein production.
We finished gluing the main body parts together and are still waiting for a reprint (one part was too big to fit inside the box). We are going to start soldering soon.
Week 1
We are doing the hardware assembly this week alongside conducting temperature and sterility experiments to test in the lab.
Week 2
We assembled the bioreactor last week and took it to the lab for initial testing. We might need to find alternative airtight sealing for the cap as the outflow is not performing how we wanted. The automating sampling portion is assembled and is working, but it needs to be attached and we have to assemble it with the rest of the bioreactor body.
Sampling wheel is almost done, but we still need to work on the peristaltic pump.
