Team:Tuebingen/Poster

PacMn - Phytochelatin Actuated Complexation of Manganese

Presented by Team Tuebingen 2020
Andreas Enkerlin¹, Lea Vogt¹, Florian Vögele¹, Elias Schreiner¹, Sophia Gaupp¹, David Kessler¹, Katja Sievert¹, Aarón Alexander Refisch Ortegoza¹, Luiselotte Rausch¹, Isabel Ernst¹, Anirudh Natarajan¹, Elisabeth Schneider¹, Benedikt Jäger¹, Patrick Schweizer², Lucas Mühling², Lukas Heumos², Lina Widerspick², Famke Bäuerle², Bastian Molitor³, Lars Angenent⁴

¹iGEM Student Team Member, ² iGEM Team Mentor, ³ iGEM Team Secondary PI, ⁴iGEM Team Primary PI

Abstract
Environmental heavy metal pollution has raised concerns for water quality and human health. In fact, it was found that due to climate change, and thus, rising groundwater temperatures in Germany, manganese(II) ion concentrations in local water resources gradually increase. Accordingly, we propose an approach for the measurement and retention of Manganese(II) ions in which we use genetically engineered E. coli as a bifunctional biosensor.
Our system contains a riboswitch, which triggers a fluorescent signal in the presence of Manganese(II) ions, in combination with induced complexion for clearance of these ions with phytochelatin proteins. Due to its sequence complexity, we modified the phytochelatin sequence in silico, and modeled the variants’ structures to subsequently evaluate their stability using Molecular Dynamics simulations. Upon successful cloning, the functionality and detection range of our system is assessed using titrated Manganese(II) ions, prior to investigating the signal kinetics and effects of cell density, as well as heavy metal retention in vivo.

Motivation

Detect & protect:
Heavy metal accumulates in the food chain poses a serious health threat to exposed people all around the world. Hence, manganese pollution of soil and water moved into the focus of science.
The detection of heavy metals such as manganese is often associated with an increased technical and structural effort. Especially in structurally weak areas, a simple and quick detection of manganese is therefore usually not feasible. As a result, in this year's iGEM project, we are developing the bifunctional biosensor PacMn: Phytochelatin-actuated complexation of Manganese. This easy-to-use biosensor detects and chelates manganese, thereby removing it from the environment. In this way, we are improving the standard of living in developing and emerging countries with the methods and resources of synthetic biology.

Educate:
The battle against the increased manganese concentration in many water and soil resources of this planet cannot be combated by simply detecting and binding manganese. To solve this global problem in a sustainable way, it is crucial to effectively combat not only the consequences but also its causes. Consequently, to raise awareness, educational work has also been a central pillar of our project.

Project Design

The heart of PacMn are two sensing elements, namely a manganese riboswitch (BBa_K902074) and a manganese responsive promoter (BBa_K902073). Mechanistically, two regulatory proteins (MntR and Fur) activate the promoter in the presence of manganese, leading to transcription. Additionally, the interaction of manganese with the riboswitch increases the ribosomal binding site accessibility and allows for translation of the co-transcribed gene; here, a FAST2-tagged synthetic phytochelatin [1]. FAST2 is a small protein tag which activates a fluorogen upon expression [2], thereby providing a manganese-dependent fluorescent signal. Phytochelatins are plant oligopeptides which bind heavy metal ions, thus resulting in detoxification [3]. Taken together, the phytochelatin under the control of the riboswitch allows for chelation of manganese ions. In the absence of manganese, the circuit should be repressed and no fluorescent signal is obtained (Fig. 1).

Figure 1:Mechanism of the bifunctional manganese biosensor. During high cellular manganese levels, the regulators MntR (yellow) and Fur activate the promoter (green), allowing for transcription. Additional binding of manganese to the riboswitch (orange) is necessary for translation and expression of the FAST2-tagged phytochelatin (blue and pink), which then captures manganese ions and produces a fluorescent signal. When little manganese is present in the cell, this regulation is suppressed and translation is stopped, resulting in no observable fluorescent signal.

To understand the effect of certain parts of our main PacMn construct (GA2), we designed three other constructs (GA1, GA3, and GA4) as controls. For these, we substituted the inducible promoter for a constitutive promoter and/or the phytochelatin for a blue chromoprotein (Fig. 2).

Figure 2: Schematic figure 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, where they are connected by Gibson Assembly.

References

Dambach M, Sandoval M, Updegrove TB, Anantharaman V, Aravind L, Waters LS et al. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element. Mol Cell 2015; 57(6):1099–109.
The Twinkle Factory. The science behind FAST and SplitFAST [cited 2020 Oct 15]
Gupta DK, Huang HG, Corpas FJ. Lead tolerance in plants: strategies for phytoremediation. Environ Sci Pollut Res Int 2013; 20(4):2150–61.
Biondo R, da Silva FA, Vicente EJ, Souza Sarkis JE, Schenberg ACG. Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environ Sci Technol 2012; 46(15):8325–32.

Experimental Workflow

Cloning

The cloning of our constructs can be divided into eight main steps:

1) Gibson-overhang PCR: Addition of complementary overhangs via PCR for assembly.

2) Restriction digestion of backbone: Linearization of the vector backbone of our constructs via restriction digestion.

3) Gibson Assembly: Assembling the fragments in vitro.

4) Transformation: Transforming E. coli with DNA from the assembly reaction.

5) Colony PCR: Preliminary screening of potential transformants by amplifying insert DNA.

6) Plasmid isolation: Extraction of plasmid DNA of potential transformants.

7) Restriction digestion: Linearization of plasmid DNA for size analysis of the entire plasmid.

8) Sequencing: Sequencing of plasmid DNA to rule out mutations within insert DNA.

Testing

We tested our constructs in four main experiments:
1) Fluorescence measurement with a plate reader:

2) Analysis of cell fluorescence and morphology via microscopy:

3) Testing the effect of chromoprotein expression on cell color:

4) Sub-cultivation experiment as a test for transgenerational plasmid stability:

Modeling

To improve the efficiency and stability of phytochelatin, we created other variants of it to see if we can improve the binding affinity of phytochelatin to different heavy metals.
As we assumed the thiol groups of cysteine to be responsible for the binding to manganese, we replaced the amino acid for histidine (due to its imidazole ring) and aspartic acid (due to a higher electronegativity). All three variants are subjected to the workflow of protein structure prediction, molecular dynamics simulation and binding site prediction depicted in Fig. 1.

Figure 1: Workflow for the bioinformatic analysis of phytochelatin and its variants.

The results of the structural prediction of the variants are depicted in Figure 2. The structure of the two mutated phytochelatins is helical and differ a lot from the structure of the original sequence. The initial instability of the Asp variant was solved by using the OPLS-AA/L force field instead of the AMBER99-SB force field. All of the structures were stable in a molecular dynamics simulation over 50 ns. This is a strong indicator for their stability when expressed in the lab.

Figure 2: Predicted 3D structure of the three phytochelatin variants. The colours go from blue for N-terminal of the protein to red for the C-terminal.

According to our Ion-Ligand binding-site prediction results we found out that contrary to our expectations, the binding to manganese in the original sequence might be mediated by glutamic acid instead of cysteine. We also detected that a wider variety of ions can be bound by the His and Asp variants in comparison to the original phytochelatin sequence.

We suggest to verify these findings in the laboratory, which was unfortunately impossible up until now due to lab space and time constraints. Furthermore, it might be useful to explore the possibilities of umbrella sampling to estimate and compare the binding energies of all variants to find out more exact information on how much the binding energies will differ between our variants. The generated data of the possible binding affinities to different metal types could allow us to create a whole new variant of phytochelatin, which strongly facilitates metal bindings.

Cloning results

Successful cloning depended on three main steps: PCR, Gibson Assembly®, and chemical transformation of E. coli. These steps were gradually optimized until we finally perfected the procedure to clone all four of our main composites containing slightly different constructs (Fig. 1) (see Design). These composites can now be easily assembled by anyone.

Figure 1: Agarose gel electrophoresis of colony PCR of GA1, GA2, GA3, and GA4 (corrected sequences for iGEM compatibility). The products were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. Desired band sizes: ca. 1800 bp (GA1), ca. 1200 bp (GA2), ca. 1600 bp (GA3), and ca. 1000 bp (GA4). Cell material of the old GA1-4 sequenced clones (containing a PstI restriction site) was used as positive control of PCR and gel electrophoresis. The negative control was the PCR master mix without template DNA. In total, 78 colonies were picked for GA1-4, and approximately 88 % of these showed a positive signal (correct band) on the gel.

Comparing the high cloning efficiency represented above by the colony PCR results with the cloning efficiency in our first attempt (Fig. 2), we can say with confidence that our cloning experience was a success. We were able to increase the efficiency by a large margin, demonstrating how important optimization is.

Figure 2: Gel electrophoresis of colony PCR from GA1 (old sequence, first attempt). The products were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. pUC19 is a negative control, where only the empty plasmid was transformed. Samples 12 and 13 are labelled, as these were at approximately the right size. After further analysis, only sample 13 was confirmed to be correct.

A major part of our optimization consisted in perfecting PCR to create fragments with the proper overhangs for Gibson Assembly. Our most troublesome fragment was F (FAST - Chromoprotein - Terminator fragment), which did not show any specific gel bands in the first attempts (Fig. 3 A). By switching to a more robust PCR kit and excluding unspecific bands through gel extraction, we finally obtained a specific gel band for fragment F with considerably improved purity (Fig. 3 B).

Figure 3 A: Gel electrophoresis of gradient PCR of fragment F. The products were separated by electrophoresis in 1 % agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. For this PCR, a high fidelity kit was used. Desired band size: ca. 1300 bp. The specific gel bands can not be clearly differentiated from the DNA smears.

Figure 3 B: Gel electrophoresis of PCR of fragments F and C after gel extraction. These products were separated by electrophoresis in 1% agarose gels. As a marker, the BenchTop 1kb DNA Ladder was used. For fragment F, a more robust PCR kit was used. Desired band size for F: ca. 1300 bp. Specific gel bands are observable for both fragments.

The last step to confirm successful cloning was sequencing. We completely sequenced the DNA of our composite parts with the Sanger method. Here, we realized that the sequences contained a PstI restriction site, which is incompatible with this year’s standards BioBrick RFC[10] and Type IIS RFC[1000]. Initially, we had excluded this restriction site in the design, but we accidentally ordered the unmodified sequences. By ordering the correct DNA sequences that contain a silent mutation in the leucine codon of the PstI restriction site, and by sticking to our optimized cloning procedures, we were able to correct this mistake efficiently (Fig. 4).

Figure 4: Alignment of Sanger sequencing results of the correct version of GA1 with the incorrect reference sequence (SnapGene). Primers utilized: Microsynth standard primers M13-40 (forward) and M13-r (reverse), and custom-made primers FAST_mid_FW and FAST_mid_RV. The reference sequence is incorrect, as it contains the illegal restriction site PstI. The sequence of GA1 aligns perfectly, except with the PstI restriction site.

Measurement Results

Fluorescence measurement with a plate reade

Goal: Assess the functionality of our systems by measuring emitted fluorescence after growth in three different manganese concentrations: 0 µM, 10 µM, and 20 µM (Fig. 1).

Figure 1: Fluorescence intensity of E. coli containing different constructs after incubation in media with different manganese concentrations. Mean values (+/- standard deviation) are shown. (A) 0 µM manganese(II) chloride. (B) 10 µM manganese(II) chloride. (C) 20 µM manganese(II) chloride.

Results: No clear distinction between the fluorescence emitted by E. coli containing our constructs and the control. Fluorescence intensity of E. coli containing manganese-inducible promoters (GA1 and GA2) did not increase with higher manganese concentrations.

Conclusion: We measured background noise generated by the auto-fluorescence of the cells.

Imaging by fluorescence microscopy

Goal: Assess fluorescence by fluorescence microscopy by comparing E. coli containing the empty pUC19 plasmid (negative control) and E. coli with our main construct (GA2) grown in media containing 10 µM manganese(II) chloride (Fig. 2).

Figure 2: Live cell imaging of cells under the fluorescence microscope. Laser settings: wavelengths 485 nm (absorbance) and 535 nm (emission). 1.5 x 630 zoom. (A) pUC19 control, 10 µM manganese(II) chloride. (B) GA2, 10 µM manganese(II) chloride.

Results: Very similar fluorescence intensity observed in both cultures.

Conclusion: We measured background noise generated by the auto-fluorescence of the cells.

Chromoprotein test

Goal: Qualitatively test the expression of the blue chromoprotein of GA1 and GA3 by streaking GA1 and GA3 (old and new versions) and pUC19 (negative control) clones on plates containing three different manganese(II) chloride concentrations: 0 µM, 10 µM, and 20 µM (Fig. 3).

Figure 3: E. coli colonies containing the constructs with the chromoprotein gene grown on plates with different manganese(II) chloride concentrations: (A) 0 µM, (B) 10 µM, (C) 20 µM.

Results: No visible blue color. No difference between our constructs and pUC19.

Conclusion:Expression of the chromoprotein gene did not occur.

Discovery: We took a closer look at our sequences again and noticed the absence of a ribosome binding site (RBS) between our riboswitch sequence and the phytochelatin/chromoprotein gene. The mistake in our design was caused by the misconception that the riboswitch sequence available in the parts registry would include an RBS downstream of the riboswitch.

Stability test

Goal: Assess the extent of transgenerational stability or toxicity of all constructs in comparison to the pUC19 plasmid control in E.coli by nine transfers of clones through non-selective media. Single colonies from a final selective plate were controlled by colony PCR (Fig. 4).

Figure 4: Gel electrophoresis of colony PCR. Samples were taken from the final selective plate compared to positive controls for GA1, GA2, GA3, GA4, and pUC19. As a marker, the BenchTop 1kb DNA Ladder was used.

Results: The targets of the PCRs were observed at the desired band sizes for all samples.

Conclusion:The presence of the plasmids without expression (no RBS) of our constructs is tolerated for nine transfers and therefore presumably does not constitute a heavy burden for the bacteria.

Implementation

Application

Schematic overview of the PacMn biosensor test. (1) A water sample is added to the PacMn biosensor system. If a certain manganese concentration is present, a tagged protein will be expressed. (2) Fluorogen molecules are added to the mix. If the tagged proteins are present, the fluorogens will bind to the tags and become fluorescent. (3) The fluorescence signal is measured quantitatively using a plate reader.

Possible clients

Private firms active in manganese (Mn) mining and processing.
Municipal wastewater treatment facilities.
The scientific community and regulatory bodies interested in the effects of Mn-species.
Consumers concerned about the quality of their drinking water.

Limitations

Genetically modified E. coli as host is controlled by strict laws to prevent unintentional release into the environment.
Sufficient Mn-sensitivity and fluorescent signal intensity for detection.
Maintaining consistent precision in quantitative measurements as variable environmental conditions may influence the biosensor.
Providing an affordable system.

Possible future improvements

Combination of the sensing capabilities with other heavy-metal-elements, such as cadmium or lead.
Development of hardware to make the system applicable in a non-lab environment.
Improvement of the Mn-binding capabilities of the system by engineering the phytochelatin to have a higher binding constant.
Transforming the biosensor system into a cell-free system.

Outlook

Including an RBS (Riboswitch): Fixing our constructs.
- Ordering corrected DNA fragments and repeating our optimized cloning procedures
- Or: Appending an RBS either downstream of the riboswitch fragment or upstream of the FAST2 fragment by PCR amplification with primers containing the RBS sequence
Intracellular manganese homeostasis: Potential for system improvement.
- Constitutive expression of the manganese importer MntH to uncouple it from the manganese homeostasis regulatory network of E. coli
- Attempt to increase manganese import by inducing cellular oxidative stress
Phytochelatin-specific experiments: Assessing whether the phytochelatin successfully binds manganese.
- Testing for “rescue” effect of phytochelatin expression in high manganese concentrations (Fig. 1)
- Investigation of phytochelatin-expressing E. coli and their presumed withdrawal of manganese from the environment
- Measurement of intracellular manganese accumulation in phytochelatin-expressing bacteria using mass spectrometry

Figure 1: Experiment to determine cell growth in different manganese(II) chloride concentrations.

iGEM Tübingen: Human Practice Projects 2020

Dr. Thomas Riedel as an expert for groundwater pollution
Dr. Thomas Riedel works at the IWW Zentrum Wasser in North Rhine Westphalia. He is an expert in many areas related to ground and drinking water, such as the analysis of organic and inorganic pollutants in groundwater and soil and the consequences of environmental change on water management. His paper “Temperature associated changes in groundwater quality” inspired us in choosing manganese as our heavy metal of interest and made us aware of the locally enhanced manganese levels in German ground water. Later on, we decided we need him as a mentor and asked for an interview. In the interview he gave us profound insights in his work as a hydrogeologist, the up- and downsides of standard analytical tools in comparison to biosensors and gave a brief overview of the mechanisms related to the locally enhanced manganese levels in German groundwater. This helped us tremendously in developing our own project.

Dr. Philipp Thiel as an expert for drylab modeling
To obtain the opinion of an expert on the drylab part of our project, we reached out to Dr. Thiel, the coordinator of the Institute for Bioinformatics and Medical Informatics at the University of Tübingen. The shape of our project was greatly influenced by his helpful comments and advice. He helped us to evaluate the time needed for certain steps and pointed out possible difficulties. We integrated this feedback by focusing on finding the structure of phytochelatin variants and analysing them with molecular dynamics simulation, while discontinuing our work on riboswitches and docking. Our reasoning was the higher abundance of modelling options for peptides, such as phytochelatins, in comparison to nucleic acids sequences, such as the riboswitch, and the shortage of tools for docking proteins with inorganic ions.

Maastricht’s MSP and Muggle Journal Inititative
This year, MSP-Maastricht’s iGEM team has started a journal initiative that gives participating iGEM Teams the opportunity to publish their work in a proceedings journal. After submission of the final versions, all participating teams could vote for their favorite article. The ones with the highest number of votes were published in the printed version of the journal, making the entire process similar to any other scientific journal. Since Tübingen is one of the main locations where a potential corona vaccine is being tested, we decided to interview the director of the University Hospital Tübingen who is also the head of the clinical trial for the vaccine: Prof. Dr. Peter Kremsner. Through this interview, we gained important insight into the current status of the trial. With the consent of Prof. Kremsner, we were happy to contribute our interview to the journal initiative. We are very proud to have received so many votes that our article is now a part of the printed version. Thus, among two others, our article is mentioned on the title page of the journal.
After receiving great comments and feedback about our Interview with Prof. Dr. Peter Kremsner about COVID-19 and vaccines studies we decided to also participate in the Muggle Journal from iGEM MSP. In this article, we focused on delivering the same content but in an easy understandable way to address a broader audience. Therefore, information about the SARS-CoV-2 virus is now also available for interested people who do not have a scientific background. Additionally, this time we designed some figures to make the understanding of the content simpler. This article will be published online and as a printed version.

iGEM Tübingen: Education Projects 2020

"The children of today hold the future of tomorrow..."

From here emerges our strong motivation to promote science education. This year, we put a lot of effort into designing a sustainable knowledge transfer concept during our Chile Class project. Over a couple of months, we taught the students the foundations of synthetic biology and introduced them to scientific careers, scientific writing, and ethics in science. The students benefited strongly from our structured and reflected approach, since the commitment of our whole team resulted in tailor-made classes according to their interests.

Acknowldgements

Attributions

Student members:
Lea Vogt, Andreas Enkerlin, Aaron Refisch, Isabel Ernst, Florian Vögele, David Kessler, Benedikt Jäger, Katja Sievert, Luiselotte Rausch, Sophia Gaupp, Anirudh Natarajan, Elias Schreiner, Elisabeth Schneider

Student advisors:
Lina Widerspick, Lukas Heumos, Famke Bäuerle

Instructors:
Lucas Mühling, Patrick Schweizer

Primary PI:
Prof. Dr. Largus T. Angenent

Secondary PI:
Dr. Bastian Molitor

Partner Institutions:
Boeckler lab and Wizenmann lab at the University of Tuebingen

iGEM Team-collaborations:
Teams Stuttgart, Waterloo, Peru, Edinburgh, Maastricht

Independent collaborators:
Technical experts: Dr. Thomas Riedel, Dr. Philip Thiel, Dr. Peter Kremsner, and Spedition Brucker, Christina Sievert, Nicole B., Andrea E., Innovation Center Tuebingen, Neckaralb Live Reutlingen