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Team:Tuebingen/Engineering

PacMn

Engineering

Wetlab

Figure 1: Engineering-Design-Cycle with details how each step was or should be tackled within
                        the wet lab work.

Figure 1: Engineering-Design-Cycle with details how each step was or should be tackled within the wet lab work.

Biological engineering is a major part of iGEM’s objectives, already contained in the acronym itself. Thus, this creative and systematic process, necessary for great projects and outcomes, is supposed to receive greater attention this year. Figure 1 gives an overview of the Engineering-Design-Cycle and how we fulfilled each step.
Research: During project finding and planning, we researched literature on heavy metal pollution and on the possibilities of binding heavy metal ions. Environmental heavy metal contamination is often man-made, stemming from our agricultural and industrial activities [1, 2]. This is not only the case in developing countries [3, 4]; areas with heavy metal contaminated soil can be found in Europe [5]. Furthermore, it was shown that rising ground water temperatures are connected to higher manganese concentrations in Germany [6]. Thus, rising temperatures caused by climate change could possibly enhance problems with heavy metal pollution. This local connection and the fact that not many iGEM projects have worked with this metal so far, led to our decision to focus on manganese. Plants, animals, and human health can be affected by manganese exposure. In plants, exposure to manganese pollution causes biomass loss. In humans, its inhalation causes neurotoxicity [7–10], and recent studies suggest that manganese-contaminated drinking water poses health risks [11]. Interestingly, plants possess a remarkable disposal mechanism for heavy metals; they produce cysteine-rich oligopeptides named phytochelatins, which form complexes with heavy metal ions, especially cadmium. The complexes are then stored in the vacuole, preventing interference with cellular biochemical processes [12]. To address heavy metal contamination, an overexpression of synthetic phytochelatins on the surface of E. coli was attempted and resulted in more accumulation of cadmium in comparison to wild-type E. coli [13].
Imagine: This led us to our project PacMn: Phytochelatin-actuated complexation of Manganese. To combat heavy metal contamination in water, methods for detection and capture of these ions are crucial. Therefore, we want to create a bifunctional biosensor that fulfills both tasks using the synthetic phytochelatin EC20 already included in the parts registry (BBa_K1321005), which has been shown to increase manganese adsorption when expressed on C. metallidurans (14). although to a lower extent than other metals. Thus, future adaptations of phytochelatin may be necessary to increase its complexing efficiency for manganese.
One of our primary aims is to have a bifunctional biosensor with a high specificity for manganese ions. Accordingly, we hypothesize that by using a riboswitch-based sensing mechanism, we will gain stronger specificity than via standard repressor / activator proteins of bacterial operons [15]. The effects that manganese-complexing will have on the functionality of the biosensor are unknown. At this point, we can only assume that complexed manganese ions in the phytochelatin will not be sensed by the riboswitch due to its high specificity for free dissolved ions [16], creating a self-regulated system in the form of a negative feedback loop. In contrast, if riboswitches could be stimulated by complexed manganese ions, an intracellular accumulation of manganese would lead to a positive feedback loop and, ultimately, a false positive signal for extracellular manganese concentrations.
Because E. coli is a model organism for cloning work, we chose it as our biosensor host. However, other microbial organisms might be more suitable for distinct extreme conditions. Thus, at a later stage, we may consider switching hosts. Naturally, we wish to create a product that adheres to biosafety regulations. Considering our work will entail the usage of genetically modified organisms (GMOs), a potential safety measurement for the application of our product would be the implementation of a filter system permeable to manganese ions and impermeable to cells.
Design: To address our questions and reduce unknown variables which would arise in a more complex system, we decided to simplify our design as much as possible. In order to study the individual components of each construct, we designed four similar constructs for comparative analysis (Table 1). Although only one type of phytochelatin was included in our constructs, other variants were also modelled by our dry lab.
Table 1: Description of all components (BioBricks) of our PacMn constructs.
Name Part ID Function
Manganese- promoter BBa_K902073 A manganese-inducible promoter for transcriptional regulation
Anderson-promoter BBa_J23102 A constitutive promoter to evaluate the effect of having and not having transcriptional regulation
Manganese- riboswitch BBa_K902074 A manganese-inducible riboswitch for translational regulation
FAST2-tag BBa_K3510000 A fluorogen-activating protein for interchangeable fluorescence
Phytochelatin BBa_K1321005 A chelator for manganese-decontamination
Chromoprotein BBa_K864401 A blue chromoprotein to evaluate the effect of phytochelatin on the sensing mechanism of the construct and to provide a direct color change for the naked eye
Double terminator BBa_B0015 A reliable double terminator
Different combinations of the seven components (Table 1) resulted in four PacMn constructs (Fig. 2). For details regarding design and function of these parts please visit the corresponding wiki page.
Figure 2: Schematic overview of the PacMn constructs. Each Gibson Assembly consisted of two fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert fragments. Here they are going to get connected by Gibson Assembly.

Figure 2: Schematic overview of the PacMn constructs. Each Gibson Assembly consisted of two fragments and a pUC19 vector backbone. The dotted line indicates the ends of the two insert fragments. Here they are going to get connected by Gibson Assembly.

Build: To assemble our constructs, we chose the Gibson Assembly® method, which is an efficient and seamless method for assembly of multiple linear DNA fragments [17]. This method relies on the addition of complementary overhangs to each DNA fragment by PCR. The overhangs of a fragment must be complementary to its adjacent fragments, and careful primer design is required, in which we succeeded with the support of the SnapGene software. The extended fragments were assembled together along with pUC19 as the vector backbone. Upon successful cloning, our constructs were subjected to measurement experiments and others.
As neither the initial PCRs nor the Gibson Assembly had worked on the first try, we had to iterate through the Build-Test-Learn-Improve cycle multiple times, before we finally obtained the correct constructs.
Test: Initial issues occurred with the PCRs to create the overhangs for Gibson Assembly. When analyzing PCR products by gel electrophoresis, we encountered several problematic results ranging from no bands, over faint bands, to many unspecific bands.
Learn: Therefore, we had to investigate ways to troubleshoot and optimize PCR. A summary can be found in Contributions.
Improve: With this knowledge we then edited the PCR protocol for each fragment and decided to include gel-extraction in our process.
Build: Once all PCRs worked and we had all fragments with the necessary overhangs, we approached Gibson Assembly.
Test: To assess successful assembly and transformation, we performed colony-PCR as an initial detection method. Plasmid DNA was then isolated from potential candidates and subjected to analytical restriction digestion with the restriction enzymes EcoRI and HindIII. Only the GA1 construct (Fig. 1) was successfully cloned on the first try.
Learn & Improve: Following the failed attempt to clone the other three constructs of the manganese project, we adjusted the vector-insert molar ratios based on the analysis of the gel images from colony-PCR.
Build & Test: With this adjustment, we were able to clone the other three main constructs (GA2-4) the second time we attempted Gibson Assembly. All positive clones were then sequenced to check for mutations. Unfortunately, we noticed that an ordering mistake was made for the FAST2I-tag. The original FAST2-tag sequence contains the restriction site for PstI, which is incompatible with the accepted iGEM standards BioBrick RFC[10] and Type IIS RFC[1000]. This issue was initially excluded with a silent mutation in the restriction site. However, the original FAST2-tag sequence was accidentally ordered instead of our modified version.
Learn: To solve the compatibility issue, we considered two options: Ordering the correct sequences and repeating the cloning work with our reliable protocols, or ordering primers for site-directed mutagenesis. Due to the late detection of the problem and the differing shipping times of primers and longer DNA fragments, we decided to order both and execute the method that was more readily available.
Build: To limit invested time and money, and considering we already had a polymerase appropriate for site-directed mutagenesis, we did not order a special kit for this. Since both orders arrived on the same day, we chose to repeat PCR and Gibson Assembly with the new FAST2-tag sequences, which was the more familiar and therefore reliable cloning method for us.
Test: To assess successful assembly and transformation, we applied the same test procedure as previously: Colony PCR, analytical restriction digestion of isolated plasmid DNA, and sequencing. Correct transformants were obtained on the first try for all four constructs. For assessment of the site-directed mutagenesis, we had planned to digest isolated plasmid DNA of potential transformants (after analysis of colony PCR) with PstI to verify if the restriction site was successfully changed or not. The samples which were not cut would have then been sequenced for final confirmation of successful mutagenesis. For our proposal on how to successfully assemble GA5 and GA6 from our vitamin B12 side project, which are composed of one more fragment than GA1-4, see Outlook.
Despite our encountered problem with the iGEM BioBrick compatibility, we continued experimenting with the already older constructs to gain a first impression of what results to expect, and to optimize the experimental set-up. By changing one nucleotide at a wobble position in the PstI restriction site, the resulting amino acid sequence is identical. Of course, codon usage is irregular, but this only provides more value by allowing comparative analysis of two constructs that are different in their nucleotide sequence yet identical in their amino acid sequence. The information collected could then serve future iGEM teams when deciding if a particular illegal sequence can be changed in a certain way that makes it compatible with the standards and comparibly functional to its original form.
The experimental results obtained from the older and also from the newly modified constructs indicated that there was a functional issue: No color change nor obvious fluorescence was observed in any case, contrary to our anticipated results. The fluorescence intensity measured from E. coli containing FAST2 was similar to the negative control, as only autofluorescence was observed. Furthermore, no amount of manganese was able to induce a color change in E. coli containing the chromoprotein.
Research: Therefore, we returned to literature research and took a closer look at our design to figure out where a problem might have occured. We quickly detected a misconception during our project design that led to these results. Originally, we thought that due to the nature of the relationship between a riboswitch and an RBS, they would have to be at a specific distance from each other and, therefore, a RBS would be included in the riboswitch sequence from the parts registry. In accordance with our presumption, we did not add an RBS in front of the FAST2 sequence, as this would have overridden the regulatory function of the riboswitch. This turned out to be an expensive mistake leading to no expression of our genes of interest.
Design & Test: At the time of realization, we were a few days away from wiki freeze. Since there was no time to order new sequences, and repeat our mastered Gibson Assembly method, we came up with a plan to at least test the functionality of our manganese-inducible promoter. Our plan was to analyze transcriptional regulation, since this was independent of the RBS. The only method available to us on such short notice was RT-PCR to analyze mRNA synthesis. We designed a qualitative experiment to determine if mRNA synthesis was being promoted by the manganese-inducible promoter of GA1 and GA2 in media containing 10 µM manganese(II) chloride. We even thought of a creative way of lysing our cells for RNA extraction (see Troubleshooting). Unfortunately, the experiment was inconclusive (see Results).
Research & Imagine: While conducting our troubleshooting literature research, we also gathered more knowledge about manganese homeostasis in E. coli. It was found that the cells reduce the expression of the manganese importer MntH in the presence of high manganese levels [18, 19]. This may interfere with our biosensor, as it would prevent a proportional response of the sensor towards extracellular manganese concentrations. In contrast, it was shown that manganese import is increased under oxidative stress [19]. Strains lacking catalase/peroxidase (Hpx-) cannot degrade hydrogen peroxide (H2O2) and accumulate H2O2 when cultured in oxic media [20]. As presented, there are multiple options for optimization of our system (see Outlook).
Conclusion: Looking back at all the roadblocks that we were able to overcome, we consider our engineering experience as successful. We experienced first-hand how unpredictable and sensitive laboratory work can be, and how beautiful it is when different minds cooperate to find solutions for our human mistakes. Like for many others, the SARS-CoV-2 pandemic was our largest roadblock. Its consequences included difficulties finding a lab and consequently our late start there in the middle of Summer, transferring to a second lab in Autumn, the limited time and space available in these labs to ensure safety in the workplace, and not being able to meet in real life to discuss early design and planning steps.
In spite of the difficult situation, we managed to clone our main composite parts by iterating through the Engineering-Design-Cycle (Fig. 1). Using the extensive toolbox synthetic biology has to offer, we overcame many issues occurring in the wet lab, especially for preparing our individual fragments for Gibson Assembly, which took time to perfect. For cloning of our composite parts, we managed to troubleshoot efficiently and can now provide reliable protocols to replicate this section of our work. By applying the Build-Test-Learn-Improve cycle throughout the course of our work, we detected the sequence mistake responsible for the shortcomings of our composite parts, and can now proceed to fix this mistake and test them again. This proves how important it is to follow this procedure in a meticulous way to implement a new system in synthetic biology.
Although we realized that our systems require a sequence adjustment to work as expected, and we lacked the time to correct this before this year’s wiki freeze, all of us are satisfied with the development of our project. Each and every one of us learned new skills and became a better scientist in a matter of months. We could not have asked for a more realistic experience to teach us how complicated and fun science can be.

Drylab

Figure 3: Engineering-Design-Cycle with details how each step was approached within the dry lab work.

Figure 3: Engineering-Design-Cycle with details how each step was approached within the dry lab work.

Research: In order to find a way to contribute to the project from a perspective of modelling, the drylab members conducted literature and database research on their own, primarily about riboswitches, phytochelatin, and their binding mechanisms to manganese and other ions. Based on this research, we assumed that the amino acid cysteine, which appears often in our phytochelatin, binds to the manganese ions through its thiol groups.
Imagine: We identified the great potential in the improvement of the binding of phytochelatin to manganese, so we decided to tackle this challenge. Additionally, we realized that the three-dimensional structure of the phytochelatin we are using is unknown. This led us to wonder how replacing cysteine with other amino acids would affect the structure of our phytochelatin and its binding to manganese.
Design: We exchanged all cysteine codons in the phytochelatin sequence, once for histidine and once for aspartic acid codons. The decision was based on their higher electronegativity in comparison to cysteine, which might strengthen the binding to manganese.
Build: The structures of the original sequence and the two modified sequences were predicted using a mixture of homology and ab initio modelling (see Model).
Test: To test the stability of our newfound structures, we needed to employ molecular dynamics modelling over 50 ns.
Learn: While the structure of the original sequence and the histidine variant were stable over the whole simulation time span, the aspartic acid variant was not. We then had to evaluate whether to discard the structure or change the model parameters. From our collaboration with Waterloo, we learned about the potential of quantum mechanical force fields such as CHARMM for better estimation of sequence stability.
Improve: After changing the forcefield, the structure of the aspartic acid variant was stable for a relatively long time of 48 ns. Through this modification, we were able to validate this structure as well.

References

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