Team:Aalto-Helsinki/Engineering
The Capsule
To ensure maximum functionality and success of GutLux, many factors were accounted for. Factors contributing to the successful engineering of the external capsule and its internal compartments are described below. The design of the capsule has been influenced by previous literature. For more information on what inspired this design, refer to Design and the Design section on the Hardware page.
The Biological Compartment: From Metabolite Concentration to Light Signal
Our biological mechanism can be separated into two parts: our proteins needed for metabolite binding and our reporter plasmid.
Preparing for Differences Between Bacteria and Yeast
Given that we worked with both prokaryotic and eukaryotic cells, there were some differences in how we built our biosensor cells, for example, incorporating different ori sites and selection markers. Furthermore, since Saccharomyces cerevisiae cells contain a nucleus, our reporter plasmid was integrated into the yeast genome so it could express our promoter containing the dioxin response element (DRE) and downstream green fluorescent protein (GFP) endogenously. How we factored for these differences is justified in the following sections.
Choice of Cloning Method
To produce our proteins the Aryl hydrocarbon Receptor (AhR), Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and Aryl hydrocarbon Receptor Interacting Protein (AIP), we designed our final plasmid to contain them all sequentially. This was done so they can be co-expressed in the whole cell to facilitate the successful detection of the metabolite. To do so, we decided to use the modular cloning method, known as MoClo. We opted for this method of cloning because it offered more autonomy and flexibility with our plasmid conformation. MoClo works so that the individual elements of a functional plasmid exist separately, and are designed in a way so subsequent plasmids can be built from these individual parts to the scientist’s desire. We developed our MoClo protocol after the RFC94 cloning system created by the BostonU iGEM team. To learn more about how we performed our cloning, refer to the Wet lab page and our Parts page for an extensive list of our individual parts.
Choice of Fluorescent Protein
Our reporter plasmid would be responsible for producing a green fluorescent protein in the presence of detected metabolite. We opted to use a superfolder GFP (sfGFP) as it produces a more intense fluorescent signal that would be easier to detect by our light dependent resistor.
Choice of Promoter
In our bacterial cells, we chose to express our proteins in our Level 1 and Level 2 plasmids under the constitutive promoter, T7. We decided on using a constitutive promoter over an inducible promoter to help increase protein yield within the bacterial cell.
In our yeast cells, we opted for three different weak promoters: POP6, RNR2, SAC6 (corresponding to AhR, ARNT, AIP, respectively). These were chosen to minimize the risk of exhausting translational machinery if we were to use strong promoters instead. The use of three different promoters was chosen since we wanted to prevent the potential of recombination, as we were working with a multigene plasmid.
For our reporter plasmid, we used the CYP1A1 promoter. This promoter, while useful because it naturally contains the DRE-element necessary for our metabolite detection, was also chosen because it is an inducible promoter. We designed our reporter plasmid with an inducible promoter in order to minimize false positive signals resulting from AhR-ARNT binding without a ligand present. We also used an inducible promoter over a constitutive promoter in the reporter plasmid in order to decrease background noise and increase chances of a functioning biosensor cell. It is possible that using a constitutive promoter could exhaust our biosensor cell and its resources quickly if it was responsible for transcribing and translating proteins from two plasmids both under a constitutive promoter.
Addition of a Purification Tag
We initially hoped to create a cell-free metabolite-detection system in order to overcome genetically modified organism (GMO) safety concerns with our biosensor. Our idea was to purify our AhR, ARNT, and AIP proteins and add them to a cell-free lysate. To aid in protein purification, a His tag was added at the N-terminus of our AhR, ARNT & AIP protein coding sequences. Unfortunately, after discussions with experts in cell-free protein expression, we decided against cell-free expression, and the purification tags in our constructs were then decidedly redundant.
Choice of Solubility Tag
Given the flexibility of MoClo, we decided to include the option of adding a glutathione s-transferase (GST) solubility tag to our proteins to prevent protein aggregation. This was a level 0 part that is listed on the Parts page. When designing our inserts, we made sure to include an enterokinase protease at the end of our insert, right before where the solubility tag would ligate to. This was included so that we could remove the GST tag if it potentially interfered with the actual protein function.
Choice of Ori Site
The ori sites of our plasmids had to be tactfully chosen given the complexity of our project. Since our bacterial engineered cells would be containing two plasmids (protein plasmid + reporter plasmid), we needed to make sure that it would be possible for the Eschericia coli cells to uptake both plasmids, not just one. To increase the probability of both plasmids being taken up and for optimal functionality within the bacterial cell, two different ori sites were chosen. For the final protein plasmid, an ori site from incompatibility group B was chosen, and an ori site from incompatibility group A was chosen for our reporter plasmid.
Yeast specific ori sites were chosen for the yeast protein plasmids as they function differently than bacterial cells. We chose the CEN/ARS ori site for our yeast protein plasmid as it is commonly used in commercial yeast expression vectors.
Alternatively, our reporter plasmid in yeast did not contain an ori site, as it was required to be integrated into the eukaryotic yeast genome. We termed this as our yeast integration plasmid. Instead of an ori site, repetitive sequences were flanked around our sequence of interest that was to be integrated into the genome.
Choice of Selection Markers
Given our multi-plasmid workflow with modular cloning, we had multiple selection markers to guarantee that our selected bacterial and yeast colonies were correctly transformed. The table below lists the antibiotic resistance/auxotrophic marker per plasmid.
Choice of Optimal Metabolite Detection
We utilized models and performed a docking analysis to examine our sensor’s sensitivity towards our chosen metabolites and to better improve specific metabolite detection. To learn more, refer to our Model page.
Design Cycle: Prototype
Design
Based on our discussions with experts and mentors, we opted to develop our prototype, which converts our biological signal to readable data, functionality-first. To test the main functionalities of our capsule’s electronic system, we wanted to be able to reach the following goals:
- Collect data from light intensity measurement at the expected wavelength area
- Send data wirelessly to a receiver device
Build
We built our prototype using Arduino Uno, a light-dependent resistor (LDR), radio frequency (RF) transceivers and a light-emitting diode (LED). We chose the LDR and LED components to mimic the expected fluorescence detection. To read more on component choices and our Arduino design, check our Hardware page.
Test
We tested our prototype with a program that simultaneously controls the LED to illuminate with fluctuating intensity, measures the intensity values with our LDR component and sends the data through radio transmittance. We were able to successfully record the intensity values as a function of time, and read them on the monitor at the receiver end. This proves that our preliminary prototype performs as expected, and can achieve the goals set at the beginning.
Learn
As a result of our prototype development, we were able to understand the basic functionality of our electronic system. This gave us the opportunity to continue on with the research of optimal components, which would work in the proposed pill-size application. Our experiments with radio frequency also provided us with insight on the type of raw data we obtain. This helps us to understand the signal we obtain from the measurement, and serves as a starting point for further signal processing planning.
Design Cycle: Plasmid Design
After determining the components described above, we worked to implement these ideas and test them out following the engineering design cycle.
Design
We spent a significant amount of time designing our plasmids to make sure they would function as desired. Also, given that we would only have 3 months in the lab, we wanted to maximize our transformation efficacy by building our plasmids correctly. After we decided to opt for modular cloning, we consulted with the 2020 Aalto-Helsinki iGEM team, as they worked with MoClo as well. Through their mentorship, we made sure to avoid some common mistakes and implemented any advice they offered during our design process.
Our bacterial Level 1 plasmids were built to contain a T7 promoter, a ribosomal binding site (RBS), the protein coding sequence (AhR, ARNT or AIP), a GST tag OR an alternative linker sequence and a T7 terminator alongside the backbone with an applicable antibiotic resistance gene. Figure 2 depicts two functional AHR Level 1 plasmids, just with or without a GST tag.
Input from our Aalto-Helsinki predecessors, staff scientist Chris Jonkergouw and postdoctoral researcher Kostantin Kogan were especially helpful during this plasmid design process, particularly when justifying our promoter choice, selection of ori sites and proofreading our final plasmid maps.
Build
After designing our plasmid maps, we began to work on the actual construction of our plasmids in the lab. We ordered our gene fragments with our individual Level 0 parts from IDT and obtained our backbones from Aalto researchers. Here we encountered our first minor wet-lab challenge -- a misunderstanding during the ordering process of our gene fragments. We constructed our inserts to be synthesized and delivered within a circular plasmid backbone. Unfortunately, we received linear fragments instead. To fix this, we had to construct our Level 0 plasmids ourselves. We opted to do this through a commercially available blunt end cloning kit from Thermo Fisher Scientific.
To assemble our Level 1’s, we performed traditional restriction digestion and ligation with type IIS enzymes. For further information on our experiments, read more on our Wet lab page.
Test
To test for the successful creation of our Level 0 and Level 1 plasmids, we digested them and performed agarose gel electrophoresis experiments.
Our Level 0 plasmids assembled relatively easily. Our gel analysis showed that the band sizes were representative of the insert size, and thus were correctly created. Figure 4 below shows the agarose gel electrophoresis images that determine our successful Level 0 parts (found in Table 2).
Our Level 1 plasmids took a little more time to assemble. We had performed restriction digestion and ligation a few times with varying gel electrophoresis results. As such, we had to troubleshoot and optimize our protocols (Figure 5).
Learn
To troubleshoot our Level 1’s, we looked at variables within the protocol that we could adjust and test out. After a few adjustments, we found optimal results with the following parameters. Further details on these adjustments can be found on the Contribution page.
Molar ratios: The original 10 fmol of plasmid DNA was not sufficient for our use, so we increased our concentrations to 50-100 fmol. Successful clones were found when we adopted a vector:insert ratio to 1:3.
Enzyme Quantities: We had to increase our enzyme volumes 2.5x more per reaction.
Digestion/Ligation Time (Enzymatic Reaction Time): This varied per reaction, some Level 1’s were formed with 25 cycles in the Thermocycler with 1.5 minutes for digestion and 3 minutes ligation and others required 30-35 cycles with 3-5 minutes for digestion and 5-7 minutes of ligation. For some Level 1’s, an additional 30 minute digestion period prior to cycling was beneficial.
After we successfully optimized the protocol for some of our Level 1’s and verified them with gel electrophoresis, we proceeded to perform gel purification experiments to extract the verified DNA.
We struggled with low yield after gel purification, despite the gel purification kits promising yields over 90%. After discussing with our Principle Investigator, Heli Viskari, we adjusted a few parameters to try and increase yield. We lowered the agarose gel percentage from 1% to 0.8%, used different purification columns, and even tried out different purification kits, but to no avail. We were eventually able to optimize our protocol to increase yield from 10% to 40-60%. To reference our optimized protocol, please refer to the Contribution page.
