Engineering

Cultural Heritage Preservation: Our inspiration

The search for our project began by defining a topic which everyone in our team was passionate about. The majority of the previous MSP-iGEM teams conducted projects centered around environmental themes. Our team decided to chart a slightly different course: using synthetic biology in the field of cultural heritage preservation.

The primary inspiration for our project came from three papers which described treatments with microorganisms in order to remove accumulated pollutants from stone surfaces of historic buildings in Rome (Alisi et al. 2021, Ranalli et al. 2021, Gioventu et al. 2011).^[1][2][3]

Another important source of inspiration was the book “ Microorganisms in the Deterioration and Preservation of Cultural Heritage"(Joseph, 2021).^[4] In particular, the chapters by Sprocati et al. (2021)^[5]and Jroundi et al. (2021)^[6] outlining the use of microorganisms for bio-cleaning and the consolidation of stone artworks inspired the basic concept of our first project idea.

Our initial project

Initially, our project aimed to engineer a microorganism with a dual function: the ability to remove black crust on stone surfaces while also being capable of filling cracks in the stone structure. This dual system would switch between these functions via an oxygen switch. In hypoxic conditions, such as the inside of a crack, the consolidation pathway would be activated leading to formation of calcite. Once the outside of the crack has been reached, the presence of oxygen would flip the switch and activate a black crust removal pathway inspired.

This idea drew inspiration from ZJU China 2022's^[7] oxygen-sensitive quorum sensing module and the metabolic pathway for black crust removal developed by iGEM Trento 2012.^[8]

However, after extensive research and brainstorming, we eventually came to the conclusion that using a single microorganism to get to the desired outcome presented significant challenges, especially taking into account the limited time due to the competition. Combining the two reactions would mean having to prevent crust removal within the cracks while restricting calcite formation outside of them. This presented significant technical challenges such as incorporating a quorum sensing module.

SublimeStone: Consolidation of limestone

Rather than attempting both biocleaning and consolidation of stone cultural heritage, we decided to shift our focus towards the consolidation of stone artworks. Particular emphasis is put on the consolidation of limestone heritage, as it is a ubiquitous building material that holds significant historical importance in our city of Maastricht.

Our goal for the consolidation mechanism was divided into two main tasks:

Efficient Crack Filling via Microbially Induced Calcite Precipitation (MICP):

We explored various microbial pathways for $CaCO_3$ precipitation. We selected a pathway that uses carbonic anhydrase as the catalyst for producing $CaCO_3$.

This presents an environmentally friendly consolidation method as it utilizes $CO_2$from the environment as a substrate and produces no toxic byproducts in contrast to other MICP pathways.

Creating a Robust Scaffold within the Cracks to Preserve Artwork's Structural Integrity:

It quickly became apparent that a robust scaffold would need to be introduced in the cracks for efficient consolidation via MICP. In absence of nucleation sites, MICP will produce amorphous calcium carbonate (ACC) which lacks the stability to withstand high mechanical stresses (Athanasiadou & Carneiro, 2021).^[9]

Our PI Erik proposed the idea of using DNA nanostructures as a scaffold, as he had prior experience with this technique. The study by Athanasiadou & Carneiro demonstrated the successful use of DNA nanostructures as scaffolds for biomineralization, highlighting their potential to offer structural support within the cracks of limestone structures.

In order to chose a specific microorganism, the team got inspired by a paper from Khaliq & Basit Ehsan (2016)^[10] evaluating the impressive performance of B. Subtilis for natural carbonate precipitation.

Calcium Carbonate Precipitation

Biomineralisation is the process by which living organisms synthesise minerals (Dhami et al., 2013)^[11]. Calcium carbonate ($CaCO_3$) precipitation is a process that naturally occurs amongst many types of bacteria, such as sulphur-reducing and urease-positive bacteria. Bio-$CaCO_3$ precipitation can proceed through two main metabolic pathways, using urease or carbonic anhydrase (CA) as the catalysts of the reaction (Chaparro-Acuña et al., 2019)^[12]. However, synthesis through urea hydrolysis produces ammonia as a byproduct, which is considered as a pollutant gas. Therefore, the CA pathway can provide a safer option for crack remediation (Chaparro-Acuña, et al. 2019).^[12:1]

CA is a ubiquitous zinc metallo-enzyme found across all kingdoms (Aggarwal et al., 2012).^[13] Microbially induced calcite precipitation occurs as a result of the enzymatic hydration of carbon dioxide ($CO_2$) to bicarbonate ions ($HCO_3\ ^-$) by CA (Smith & Kerry, 2000).^[14] This process creates alkaline conditions which increase the solubility of $Ca^{2+}$ ions trapped on the extracellular matrix (EPS) of bacterial cells, which readily bond to $HCO_3\ ^-$ , facilitating the formation of calcium carbonate crystals (Anbu, et al. 2016).^[15]

Thermostable CA’s from extremophilic organisms have recently shown to have a high efficiency due to their ability to retain a high $k_{cat}/K_m$ under fluctuating temperatures and varying pH (Hsieh et al., 2021).^[16] The enzymatic efficacy from CA’s expressed in a variety of organisms was analyzed. After this preliminary research it was determined that currently, the fastest known CA is derived from the extremophile Sulfurihydrogenibium azorense, termed α-SazCA (De Simone et al., 2015).^[17] Remarkably, α-SazCA ($k_{cat}/K_m$ = 3.5 × 108 M−1 s−1) demonstrated 2.33-fold higher catalytic activity in the $CO_2$hydration reaction compared to hCA II, which had long been regarded as the most proficient catalyst within this enzyme family. Additionally, α-SazCA displays low susceptibility to inhibition by anionic compounds commonly found on limestone buildings, such as bicarbonate, sulphates, and nitrates (De Luca et al.,* *2013).^[18]

Organism of choice

Escherichia Coli was chosen as the bacterial strain of choice for this project’s the modules. Nonetheless, before setting it as the final choice, the team explored various possibilities, one of which was B. subtilis , which was used in other studies such as in Mechanisms of Biomineralization by Bacillus subtilis and Characteristics of the Biominerals performed by Han et al. (2019).^[19] The article  describes the biomineralization of calcium carbonate, making it an option of  great interest for the purposes of the project. B. subtilis is an aerobic, gram positive bacterium that has an excellent protein secretion ability, with growth time approximately similar to E. Coli.

E. Coli was the other candidate for our chassis. The research by Zhu et al. (2022)^[20], which inspired our biomineralization SazCA module, used the E.coli BL21 (DE3) strain to express the protein. This competent strain has also been shown to have effective expression of membrane proteins.

Given the possibility of both organisms to work well with our a model, the team was torn in the chassis of choice. However, as the SublimeStone initiative consists of more than one module, the compatibility of the organism with the mechanism of DNA origami production also had to be assessed. E. coli has been successfully used for bioproduction of DNA origami many times. Studies have shown that the bacterium can be used as the chassis for the helper plasmid-phagemid system, involved in ssDNA production, which would then be isolated, prepared and ready for running self-assembly reactions to produce the desired shape of the origami (Praetorius et al. 2017).^[21]

After conducting further research and comparing the capabilities of these organisms, it was decided that E. coli  would be the better fit for our project. For the project, two strains were used: BL21 (DE3) was used for the biomineralization SazCA module, and DH5ɑ was used for the DNA origami module. Different strains were used to accommodate the plasmids ordered. Both strains exhibit similar growth conditions and are not competitive when co-cultivated within the same system.

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To achieve this, we decided to separate our engineering into three modules.

Engineering Cycles Overview

Module 1: SazCA-INPN Membrane Display Module (SIMD)

This module consists of engineering BL21 DE3 E. coli that can express the SazCA enzyme on the surface of the bacterial outer membrane.

Enhancing SazCA Activity with Surface Display

SazCA, derived from the thermophilic bacterium Sulfurihydrogenibium azorense, is the fastest known carbonic anhydrase to date, boasting a $k_{cat}/K_m$ value of 3.5 × 108 M−1 s−1 (De Simone et al, 2015; De Luca et al., 2013).^[17:1][18:1] SazCA facilitates the hydration of carbon dioxide to bicarbonate and protons, creating alkaline conditions that aid the formation of calcium carbonate crystals on the extracellular matrix (EPS) of bacterial cells (Fig. 1) (Anbu, et al., 2016).^[15:1]

$$CO_2 + H_2O \leftrightarrow HCO_3\ ^- + H^+ \newline \newline$$

$$OH^- + HCO_3\ ^- \rightarrow CO_3\ ^{2-} + H_2O \newline$$

$$Ca^{2+} + CO_3\ ^{2-} \rightarrow CaCO_3$$

Figure 1

The reversible $CO_2$ hydration reaction catalysed by SazCA. In presence of $Ca^{2+}$, $CaCO_3$ is formed.

Currently, the intracellular microbial expression of carbonic anhydrase (CA) is well characterised; however, its practical utilisation has considerable drawbacks. $CO_2$ and $HCO_3\ ^-$ are small molecules capable of diffusing in and out of cells, yet the intracellular activity of CA is significantly limited by the permeability of the cell membrane, as it restricts both the amount of substrate available for catalysis and the subsequent secretion of the product, reducing overall enzymatic efficiency (Jo et al., 2013).^[22]

Our proposed strategy is to express the SazCA enzyme on the surface of the bacterial cell, directly exposing it to extracellular concentrations of $CO_2$, and bypassing cellular secretion of bicarbonate ions, further enhancing the enzymatic activity of the fastest known carbonic anhydrase to date. The design for our extracellular display of the SazCA enzyme was derived from the work by Zhu et al. (2022).^[20:1]

Construct Overview

To enhance enzymatic efficiency, this module expresses the SazCA enzyme as a fusion protein on the cell surface of E. coli. This module is based on the findings of Zhu et al. (2022)^[20:2], wherein a membrane fusion protein was designed to showcase SazCA on the surface of E. coli cells. This is achieved by linking the E. coli codon-optimized SazCA enzyme (BBa_K4665120) to the integral membrane protein INPN (BBa_K4665001) using a flexible GGGGS linker (BBa_K2549053). The resulting membrane fusion protein is shown in Fig. x and will be referred to as our SazCA-INPN Membrane Display module (SIMD). The SIMD module is constructed from three main components:

1) Ice nucleation protein N-terminal (INPN): This is the N-terminal of ice nucleation protein which will be embedded into the E. coli cell membrane. The sequence coding for the INPN is preceded by a pelB leader sequence as its expression promotes the secretion of the protein via the Sec pathway whilst avoiding hydrolysis by cytoplasmic proteases that might lower the quantity of proteins on the cell’s surface (Mergulhao et al., 2005).^[7:1] By attaching the pelB signal peptide in front of the INP protein, the fusion protein will be directed towards the bacterial periplasm where it will be anchored in the cell membrane (Singh et al., 2013).^[8:1] The INPN sequence is followed by two front-end sub-repeat sequences important for the stability of the fusion protein (Zhu et al., 2022).^[8:2]

2) GGGGS linker: The GGGGS flexible linker is composed of a sequence of 4 glycine repeats followed by a serine amino acid. This flexible linker is used to connect the N-terminal of the INP to the carbonic anhydrase which creates an elongated fusion mode that allows for optimal carbonic anhydrase stability (Hartmann et al., 2022; Zhu et al., 2022).^[9:1][6:1]

Figure 2

Visual representation of the SIMD fusion protein, taken from Zhu et al. (2022).^[6:2]

3) SazCA: This sequence codes for the carbonic anhydrase derived from Sulfurihydrogenibium azorense (SazCA). This sequence has been codon optimised for E. coli. The SazCA coding sequence is followed by a His-tag which facilitates the purification and detection of the fusion protein.

Plasmid Design

The SIMD module plasmid designed for our $CaCO_3$-producing E. coli strain is based on the work of Zhu et al. (2022).^[20:3] The sequence coding for the SazCA-INPN Membrane Display module is constructed of our three insert sequences (INPN, GGGS linker and SazCA), which were designed with complementary BsaI site overhangs to allow for Golden Gate Assembly cloning.

The SIMD Module plasmid was constructed using a pET-39b(+) backbone vector which has several features that make it a suitable backbone for the recombinant plasmid. It includes a T7 promoter with a lac operator, enabling the selective expression of our fusion protein when incubated with Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Du et al., 2021).^[23] Additionally, the pET-39b(+) vector possesses a kanamycin resistance gene which does not contain any internal BsaI sites that might interfere with the assembly of the recombinant plasmid via Golden Gate Assembly. The kanamycin resistance gene allows for the positive selection of E. coli cells which have successfully incorporated the recombinant plasmid by their ability to grow on a growth medium containing kanamycin.

Figure 3

SnapGene design of our SazCA-INPN membrane display module.

Module 2: Octahedron Lattice via DNA Origami

This module utilizes DH5α E. coli for the production of long ssDNA strands, which can subsequently be folded into DNA origami structures through the addition of short complementary ssDNA known as staples. These staples facilitate the folding of the ssDNA scaffold into an octahedron structure through complementary base-pairing. These octahedron structures contain protruding sticky ends at their vertices, allowing them to interlock and form a scaffold comprised of the DNA nanostructures. The DNA origami scaffold is the intended mineralization scaffold for our Module 1.

Any chemical process is characterized by the speed at which the reaction takes place. In many instances, the presence of a catalyst to accelerate the reaction is required in order for the chemical process to take place.

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In the case of biomineralization, the presence of nucleation site is essential to ensure the growth of crystal.

Nucleation Sites

A nucleation site is a a specific location at which the chemical process of nucleation takes places. Nucleation is the process in which a particular material undergoes a phase transition. In our case, the nucleation describes the change of state of liquid bicarbonate ions into limestone crystal. To address this, we investigated how we could develop and create a good nucleation site using synthetic biology methods.

Octahedrons via DNA Origami

DNA has been shown to enhance the biomineralization of $CaCO_3$ (Ivanova et al. 2023).^[30:5][31:4] As such, we researched within the field of DNA origami to find a suitable shape that could act as the nucleation site.

Multiple research papers have already been published containing DNA origami designs with custom functions. Our choice fell on a symmetric shape since the process of mineralization should happen evenly across the nucleation site. The octahedron offers a good balance between simplicity and mechanical strength.

ChimeraX render of the DNA-origami Octhahedron

DNA has been shown to enhance the biomineralization of $CaCO_3$ (Ivanova et al. 2023).^[30:6][31:5] As such, we researched within the field of DNA origami to find a suitable shape that could act as the nucleation site.

Multiple research papers have already been published containing DNA origami designs with custom functions. Our choice fell on a symmetric shape since the process of mineralization should happen evenly across the nucleation site. The octahedron offers a good balance between simplicity and mechanical strength.

Did you know?

Octahedron edges are solely triangles, a shape known to engineers to be mechanically the strongest.

A dual-plasmid system

The first challenge in the production of DNA origami is the synthesis of single stranded (ssDNA) scaffolds. As such, we investigated possible methods to create the scaffold.

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Due to this improvement, we decided to use the helper plasmid-phagemid system.

The Helper Plasmid: HP17_KO7 (Dietz, Addgene)

Contains the coding parts of the M13KO7 helper phage which have been cloned into a pSC101 backbone carrying a kanamycin resistance gene (Praetorius et al., 2017).^[21:1] The M13 origin of replication has been entirely removed, leading to the absence of phage production. This plasmid provides all genes necessary for the ssDNA production, but cannot initiate ssDNA production in absence of a phagemid containing the necessary M13 regulatory sequences.

The pScaf phagemid: pScaf_3024.1 (Douglas, Addgene)

The phagemid used in this project was developed by Nafisi et al. (2018).[^44] It was designed to allow for easy incorporation of a custom 3024 bp scaffold sequence tailored for single-stranded DNA (ssDNA) production. However, for our project, we opted to keep the scaffold sequence that was already present in the pScaf plasmid. The staple sequences were then designed based on this pre-existing sequence and the specific structure we desired for the DNA origami. As mentioned previously, the helper plasmid contains the regulatory sequences necessary to initiate ssDNA production.

Did you know?

Historically, the genome of the M13 bacteriophage was used as the backbone for any DNA origami design. You can still find traces of the genome in DNA origami design to this day!

Extending into a lattice

As the scale of a single octahedron is around 40-50 nm, these nucleation sites are small. To achieve higher strength of the limestone, larger nucleation sites are required. As such, we design an extension to our DNA origami module that allows for larger nano-structures to form.

To reduce the complexity of the construct, this nano-structure has to be as simple as possible. As such, a self assembling lattice of octahedrons was found to be an ideal candidate.

Wang et al. (2021)^[32:3] constructed an octahedron lattice with 2 modes. This allowed them to build a larger nano-structure of around 2 $\mu m$. The underpinning concept behind the construction of such lattice is the concept known as sticky-Ends.

Sticky-Ends

A sticky-end is defined as a short single-stranded piece of DNA that is composed of three parts.

• The first part is essentially one of the existing staples present in the model. This part binds to a specific part of the scaffold to allow the sticky ends to bind to the existing structure.

• The second part is a variable length repeat of T bases that can be designed to control the spacing between unit cells of the lattice.

• The third part is a short sequence of 10 bases that are designed to be complementary across vertices. This part is the most important, as one sticky end present in one octahedron needs to be complementary to the sticky end located on a neighbouring octahedron.

From our octahedron model, the design choice was to introduce 4 sticky ends at each vertices of the shape. The variable length repeat was initially chosen at 15 bases. The complementary part was 10 bases long and was generated computationally.

The algorithm to generate the complementary sticky-ends

As there were no known tools to generate the sticky-ends, we designed an algorithm to generate these sequences. The motivation was to generate these sequences based on an given PDB model. The main challenge behind generating these sequences is the fact that our model contains 72 different staples. Since our design involves extending some staples that are generated computationally, we wanted to identify and generate these sequences computationally as an extension to the workflow.

Info

For more information, checkout the model page of our wiki!

Future Design Improvements

Future improvements that we would like to implement when repeating the folding experiments include:

• Investigating a wider range of temperature gradients, specifically comparing the effects of slow and rapid cooling on the quality and yield of DNA origami folding.

• Implementing additional inactivation of T7 exonuclease using proteinase K. While our experiment followed the manufacturer's protocol from New England Biolabs, which uses the addition of 11 mM EDTA to inactivate T7 exonuclease, Noteborn et al. (2021)^[43:1] successfully used proteinase K for T7 exonuclease inactivation, resulting in improved folding outcomes compared to mixtures with active T7 exonuclease.

• After the folding experiment, performing PEG precipitation for the purification and concentration of the target DNA structures. This method, based on Wagenbauer et al. (2017)^[44:3], concentrates DNA origami samples by depleting high-molecular-weight species through the presence of PEG. As a result, it effectively separates the DNA origami from staple strands (Stahl et al., 2014).^[47] Concentrating the DNA origami scaffold can provide more distinct results for gel electrophoresis and subsequent TEM/SEM, reducing background noise caused by the presence of excess staples in the solution.

• Due to time restrictions, performing the folding experiments on bacterially synthesized ssDNA could not be achieved. Extraction methods should be employed to isolate the ssDNA (Behler et al. 2022; Nafisi et al., 2018).^[42:4][40:1] Folding of the bacterially produced ssDNA should be compared with the in vitro assembly products.

References

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