Rational Control of Calcium Carbonate Precipitation by Engineered

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Rational Control of Calcium Carbonate Precipitation by Engineered Escherichia coli Liya Liang, Chelsea M. Heveran, Rongming Liu, Ryan T. Gill, Aparna Nagarajan, Jeffrey Cameron, Mija Helena Hubler, Wil V. Srubar III, and Sherri Michelle Cook ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00194 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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ACS Synthetic Biology

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Rational Control of Calcium Carbonate Precipitation by

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Engineered Escherichia coli

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Liya Liang1, Chelsea Heveran2, Rongming Liu1, Ryan T. Gill1,4, Aparna Nagarajan1, Jeffrey

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Cameron1,3,5, Mija Hubler,2 Wil V. Srubar III,2 Sherri M. Cook*,2

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1Renewable

and Sustainable Energy Institute (RASEI), 2Department of Civil, Environmental, and Architectural

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Engineering, 3Department of Chemistry and Biochemistry, 4Department of Chemical and Biological Engineering,

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University of Colorado Boulder, Boulder, CO 80303, United States, 5National Renewable Energy Laboratory,

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Golden CO 80401, United States

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Abstract

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Ureolytic bacteria (e.g., Sporosarcina pasteurii) can produce calcium carbonate (CaCO3).

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Tailoring the size and shape of biogenic CaCO3 may increase the range of useful applications for

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these crystals. However, wild type Sporosarcina pasteurii is difficult to genetically engineer,

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limiting control of the organism and its crystal precipitates. Therefore, we designed, constructed,

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and compared different urease operons and expression levels for CaCO3 production in engineered

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Escherichia coli strains. We quantified urease expression and calcium uptake and characterized

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CaCO3 crystal phase and morphology for 13 engineered strains. There was a weak relationship

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between urease expression and crystal size, suggesting that genes surrounding the urease gene

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cluster affect crystal size. However, when evaluating strains with a wider range of urease

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expression levels, there was a negative relationship between urease activity and polycrystal size.

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The resulting range of crystal morphologies created by the rationally-designed strains

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demonstrates the potential for controlling biogenic CaCO3 precipitation.

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KEYWORDS: CaCO3 precipitation, MICP, Rational control, Urease, Escherichia coli Introduction

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Biologically sourced structural materials from living things (i.e., fungi, plants, bacteria),

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such as biopolymers and microbial carbonates,1–3 are being manufactured to reduce energy and

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waste associated with traditional manufacturing approaches. Specifically, microbially induced

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calcium carbonate (CaCO3) precipitation (MICP), a process where an organism creates a local

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microenvironment with conditions that permit precipitation of carbonate minerals,4 has attracted

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considerable interest in the last 20 years due to its numerous applications, such as soil stabilization,

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concrete remediation, and manufacture of bulk-scale materials.5 There are four main groups of

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microorganisms involved in the process: (i) photosynthetic organisms such as cyanobacteria and

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algae, (ii) sulfate reducing bacteria responsible for dissimilatory reduction of sulfates, (iii)

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organisms utilizing organic acids, and (iv) organisms that are involved in the nitrogen cycle either

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by ammoniﬁcation of amino acids/nitrate reduction or hydrolysis of urea.6–8 Recently, CaCO3-

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precipitating bacteria have been shown to bind sand particles to produce brick-like products.5,9–11

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Biogenic precipitation of CaCO3 via urea hydrolysis is one of several mechanisms by

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which MICP is accomplished. Ureolytic organisms, e.g., Sporosarcina pasteurii (S. pasteurii), are

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efficient at precipitating high amounts of CaCO3.12 Stocks-Fischer et al. (1999) showed that during

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microbial urease (urea amidohydrolase; EC 3.5.1.5) activity, 1 mol of urea is hydrolyzed

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intracellularly to 1 mol of ammonia and 1 mol of carbamic acid, which spontaneously hydrolyzes

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to form an additional 1 mol of ammonia and carbonic acid. These products equilibrate in water to

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form bicarbonate, ammonium, and hydroxide ions, which increases the pH.6 Urease influences the

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chemical process associated with the formation of biominerals through four different parameters7

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including pH, dissolved inorganic carbon (DIC) concentration, calcium concentration, and the

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availability of nucleation sites. The first three parameters influence the carbonate ion concentration

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(i.e., saturation state), while the last parameter (i.e., availability of nucleation sites on cells) is very

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important for stable and continuous CaCO3 formation.5,6 All of these parameters greatly affect

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either the ureolytic activity or CaCO3 crystal formation.

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While ureolytic microorganisms have been utilized for years to precipitate CaCO3,13–15

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control over this mechanism and over these organisms is challenging and substantially limits the

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ability to control properties of mineral precipitates. S. pasteurii, a soil microorganism producing

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substantial quantities of urease, has been utilized for CaCO3 biomineralization in such

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conditions.5,9–11,16,17 However, the genome of S. pasteurii is not completely understood, and

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genetic tools are not well-developed. Thus, it is challenging to engineer S. pasteurii to control

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crystal size and morphology, which limits its application for the synthesis of biogenic minerals.

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By contrast, many studies have focused on engineering Escherichia coli, which has a quick growth

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rate and extensive tools developed for its genetic manipulation, allowing greater rational control

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of CaCO3 precipitation by engineered E. coli. Bachmeier et al. studied utilizing Escherichia coli

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HB101 containing pBU11, which encodes urease gene cluster from S. pasteurii for CaCO3


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precipitation,18 and showed the potential for the rational control of CaCO3 precipitation using

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engineered E. coli.

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In this study, we designed and constructed strains with different urease operons (i.e., gene

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sequences) and strains with single (integration), medium, and high copy number plasmids to

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evaluate for CaCO3 production in engineered Escherichia coli strains. We compared urease

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expression and calcium uptake for each of the 13 constructed strains. We additionally

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characterized CaCO3 crystal phase and morphology, both in liquid solution and in solid (i.e., sand)

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cultures, for each of the engineered strains and related these to urease activities and calcium

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changes. Finally, we use this novel data to discuss the key outcomes for the rational control of

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CaCO3 precipitation process, including CaCO3 crystal phase, crystal size, and microbial

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productivity.

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Results and discussion

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The construction and verification of urease expression operons in E. coli

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E. coli was engineered to include urease operons to allow for CaCO3 precipitation (Figure

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1a). We constructed several variant urease operons to evaluate the effect of changes in expression

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on CaCO3 precipitation by directly amplifying the urease gene cluster from S. pasteurii ATCC

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11859, a wild type strain used for CaCO3 precipitation, constructing plasmids (Table 1), and then

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transforming all of the constructed plasmids into HB101 (Tables 1 and 2). We then compared the

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urease activities and the capability of CaCO3 production in all the strains (Figures 1e and 1f).The

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first engineered strain evaluated was HB101/pBU11.

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Compared to a control, the wild type strain HB101 without urease gene cluster, and to a

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baseline MICP organism, S. pasteurii ATCC 11859, the strain HB101/pBU11 had the highest

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urease activity. In contrast, the wild type strain HB101 (i.e., E. coli control) did not have any urease

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activity (Figure 1b). This confirmed that the plasmid and gene transformation was successful.

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Furthermore, Raman spectroscopy showed that the engineered strain HB101/pBU11 produced

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CaCO3 (only the calcite phase was detected) (Figure S1), which was reflected in the increased

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calcium uptake rate (Figure 1c). The wild type strain HB101 did not produce CaCO3 and no soluble

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calcium was depleted from the culture solution. This shows that the calcium uptake rate (i.e., the

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loss of soluble calcium from solution) can be used to characterize and gain insight into CaCO3

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productivity.



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Overall, these observations confirm that urease activity allows for MICP. However, it is

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uncertain how urease activity or calcium uptake impact CaCO3 production, such as crystal size

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and morphology. Therefore, we wanted to evaluate the relationship between urease activity,

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calcium uptake, and CaCO3 morphology. First, because no detailed sequences have been published

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previously for pBU11, we sequenced the whole plasmid. We found that the urease gene cluster

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(5.3 kbp same as the sequence in pBR322-Ure) had a total of 12.0 kbp gene fragments. There were

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2.7 kbp and 4.0 kbp gene sequences on both sides of the urease gene cluster (Figure 1d). Since

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these fragments or their products may affect growth, urease activity, or CaCO3 precipitation or

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morphology, we evaluated the performance of different pBU11 deletion variants.

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The effect of genes outside the urease gene cluster on CaCO3 production in engineered E.

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coli

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To further investigate the functions of these fragments, we completed protein sequence

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alignments and predicted another 4 genes (rhaTs, rhaTi, yjgF, and phoE) besides the urease gene

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cluster (Figure 1d). Then, we investigated the effects of the four genes by individual gene deletions

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(Figure 1d, Tables 1 and 2). We tested the cell growth, urease activity, and CaCO3 production and

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morphology of the deletion variants.

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The cell growth of HB101/pBU11 was the lowest of all the strains, whereas the cell growth

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of HB101/pBR322-Ure was the highest (Figure S2). All the other deletion variants had similar cell

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growth. Urease activity and calcium uptake rate, though, were different for each strain (Figures 1e

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and 1f), suggesting that they were affected by these genes or their products. For example, after 2

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h, both the variants HB101/pBU11-rhaTs and HB101/pBU11-rhaTi had the highest calcium uptake

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rates, which were similar to the baseline strain S. pasteurii ATCC 11859 (Figure 1f). The rhaTs

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and rhaTi genes encode the L-rhamnose-H+ transport proteins, which are also the members of the

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drug/metabolite transporter (DMT),19, 20 and deletion of either gene increased the calcium uptake

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rate. The strain

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lowest calcium uptake rate. The yjgF gene encodes endoribonuclease L-PSP, which has a

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conserved domain that is similar in structure to chorismate mutase. The chorismate mutase (CM,

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EC:5.4.99.5) is an enzyme of the aromatic amino acid biosynthetic pathway that catalyzes the

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reaction at the branch point of the pathway leading to L-phenylalanine and L-tyrosine production.

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To further evaluate this, we constructed the plasmid pBU11-yjgF-ure, which only contains the

HB101/pBU11-△yjgF had the


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yjgF gene and urease gene cluster. The calcium uptake rate in a strain with this plasmid,

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HB101/pBU11-yjgF-ure, was higher than rates from strains without the yjgF gene

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(HB101/pBU11-△yjgF and HB101/pBR322-Ure). These yjgF gene results suggests that aromatic

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amino acid metabolism may affect CaCO3 production.

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When investigating the relationship between calcium uptake and urease activity, the results

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showed that there was no linear relation between these two parameters (Figure 2a). For example,

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the strains HB101/pBU11-△rhaTi and HB101/pBU11-△rhaTs had the highest calcium uptake rates

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but the strain HB101/pBU11- △ rhaTi had the lowest urease activity whereas HB101/pBU11- △

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rhaTs had the highest urease activity. In addition, the wild type S. pasteurii still had the highest

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calcium uptake rate of all the deletion variants although its urease activity was average compared

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to the E. coli deletion variants. These results suggest that CaCO3 production is controlled by more

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than simply urease activity and that the genes outside the urease gene cluster also influenced

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CaCO3 productivity and urease activity.

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The effect of engineered E. coli strains on CaCO3 phase and morphology

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Calcium carbonate precipitation was studied for all seven engineered E. coli deletion

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strains as well as S. pasteurii ATCC 11859 under two conditions: flask culture and column culture

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with sand. For flask cultures, CaCO3 crystals were studied at four time points (2, 4, 8, and 18 h).

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Raman spectroscopy indicated that calcite was precipitated at all time points for all strains (Figure

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S1). Additional polymorphs of CaCO3 may be present, but Raman spectroscopy did not enable

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analysis of the entire sample.

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Crystal nucleation and growth is affected by solution saturation chemistry. Because

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calcium uptake rate, and therefore precipitation kinetics, differed amongst the deletion variants,

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we used SEM to evaluate whether these strains produced a range of sizes and morphologies. Of

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note, the CaCO3 structures studied were generally polycrystals (assemblies of smaller crystallites

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into a larger structure). Crystals precipitated by all strains in flask culture had a distribution of

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crystal sizes, yet only a weak relationship emerged between measured urease activity and

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polycrystal size. SEM at 18 h showed that the three engineered E. coli strains with the lowest

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urease activities each produced large polycrystals (Figure 2b, Group A). For example,

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HB101/pBU11-△phoE and HB101/pBU11-△rhaTi had the lowest urease activities and produced



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crystals that were relatively large. By contrast, strains with higher urease activities did not

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consistently produce large polycrystals (Figure 2b, Group B). For example, HB101/pBU11-ΔrhaTs

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and HB101-pBU11 had the highest urease activities and produced small crystals that were less

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frequently incorporated into larger polycrystals. However, there was not a simple relationship

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between urease activity and crystal size, consistent with the poor relationship between urease

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activity and calcium uptake. This poor relationship suggests that the deletions in the urease gene

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cluster may influence other aspects of CaCO3 nucleation and growth.

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In the presence of sand, S. pasteurii ATCC 11859 had the highest calcium uptake rate and

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formed many large faceted polycrystals with abundant cell castings (i.e., microbial imprints). All

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engineered E. coli strains also produced CaCO3 in the presence of sand. Although crystals are

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difficult to measure in sand, and it was not possible to survey the entire sand sample, our sample

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of SEM images suggest that HB101/pBU11-△rhaTs (highest urease activity) produced the fewest

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crystals, whereas HB101/pBU11-△rhaTi (lowest urease activity) produced the most abundant

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crystals (Figure S3). The engineered E. coli strains produced CaCO3 crystal structures that ranged

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from round mesocrystals to faceted polycrystals (Figure S4). Most strains produced a combination

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of these crystal morphologies. Overall, as with S. pasteurii, all engineered E. coli strains formed

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CaCO3 in both flask and sand cultures and exhibited evidence of cell casting on the surface of

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polycrystals that were formed in the presence of sand (Figure S3). More importantly, our

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constructed E. coli strains achieved different crystal sizes and morphologies, showing the potential

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for engineered strains to produce biogenic minerals tailored for different applications.

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CaCO3 production using strains with different urease expression levels

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We sought to understand if there could be a stronger relationship between urease activity

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and crystal size if there was a larger range in urease activity. Specifically, because gene expression

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levels depend on codon usage bias, promoter and RBS strength, and plasmid copy numbers,21–23

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we evaluated the impact of different urease expression levels (i.e., different copy numbers of the

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same urease gene) on CaCO3 production. First, we compared the different strains to identify one

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to evaluate in more detail. The comparison showed that strain HB101/pBU11, with the most

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complex urease pathway sequence, had a higher urease activity than strain HB101/pBR322-Ure,

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with the simplest urease pathway sequence. Since urease activity is dependent on solution pH,6 we

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evaluated the impact of pH on both strains. For all pH values (from 5 to 8), the strain


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HB101/pBR322-Ure had higher cell growth (Figure S5b) but lower urease activity (Figure S5a),

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although urease activity between the two strains was more similar at pH 8. For both, viability

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became an issue above pH 8; for certain applications, improved viability should be an area of

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future research. Because strain HB101/pBR322-Ure had a higher cell growth and simpler urease

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pathway sequence and because urease activity did not strongly correlate with crystal size (Figure

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2), we also evaluated this strain during subsequent rational design experiments.

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We cloned the pBR322 plasmid urease gene cluster into pRS426, a high-copy-number

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plasmid, to construct HB101/pRS426-Ure (Tables 1 and 2). Then, we integrated the urease gene

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cluster into HB101 to construct the strain HB101-Ure-integration (Figure S6; Tables 1 and 2) using

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the same CRISPR-based integration strategy as reported in Bassalo et al.,24 which demonstrated

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that safe-site 9 (ss9) of E. coli BW25113 allows for high expression of heterologous genes. Raman

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spectroscopy indicated that calcite was precipitated by the single- (integration), medium-, and

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high-copy-number plasmid strains (Figure S1). Among variants with different urease expression

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levels, the strain HB101/pRS426-Ure, with the high-copy-number plasmid, had the highest urease

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activity and calcium uptake rate, while HB101-Ure-integration, with only one-copy-number of the

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urease gene cluster, had the lowest for both (Figure 3b). We also tested these strains under different

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pH values. The urease activities of all the strains increased from pH 5 to 8 (Figure S7), similar to

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a previous study6. For the calcium uptake rate, the strain S. pasteurii had the highest uptake rates

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under pH 6 to 8 (Figure S8). However, the strains HB101-pBR426-Ure and HB101-pBR322-Ure

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had higher calcium uptake rates than S. pasteurii under pH 5.

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Given this large improvement of urease activity and calcium uptake with increased gene

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expression (i.e., copy number), we used the RBS Calculator25 and constructed new operons with

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two strong constitutive promoters (J23119 and ProD) and three synthetic RBS sequences (~10,000

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au, 5,000 au, and 1,000 au of translation initiation rates) (Figure 3a). In contrast, these synthetic

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RBS strains had much lower urease activities and calcium uptake rates than S. pasteurii ATCC

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11859, HB101/pBR322-Ure, or HB101/pRS426-Ure (Figures 3b and 3c). Further, only one of the

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synthetic RBS strains, HB101/pRS426-Ure-RBS5000, produced CaCO3 (calcite and vaterite)

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(Figure S1). To investigate why, we tested the natural RBS translation initiation rates, which were

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found to be 3,792 au, 15, 425 au, 4,358 au, 12, 201 au, 2.1 au, 4,006 au, and 49 au for genes ureA,

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ureB, ureC, ureE, ureF, ureG, and ureD, respectively (Figure 3d).

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Overall, the synthetic RBS strains all had the same translation initiation rates, but the rates



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were very different between genes for the natural RBS. For example, the natural RBS translation

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initiation rates of ureB and ureE were much higher than other genes, and the translation initiation

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rates of ureF and ureD were much lower. Following the trend of natural RBS, we then

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reconstructed the urease synthetic pathway using the synthetic RBS with “optimized” translation

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initiation rates of 5,000 au, 10,000 au, 5,000 au, 10,000 au, 1,000 au, 5,000 au, and 1,000 au for

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genes for ureA, ureB, ureC, ureE, ureF, ureG, and ureD, respectively, to create a new strain,

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HB101/pRS426-Ure-N (Table S1).The optimized synthetic RBS in strain HB101/pRS426-Ure-N

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had urease activity that was 3-times higher than the non-optimized synthetic RBS strain with the

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highest activity, HB101/pRS426-Ure-RBS5000 (Figure 3b). From Raman spectroscopy,

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HB101/pRS426-Ure-N also produced the CaCO3 polymorphs calcite and vaterite (Figure S1). This

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result suggests that rational design can lead to significantly better results and performance if strains

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with synthetic RBS sequences are constructed to reflect the relative differences between each

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gene’s natural RBS translation initiation rates.

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The largest difference in urease activity and calcium uptake between strains was due to

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copy number (Figure 3). Therefore, we evaluated CaCO3 crystals with SEM for the single-

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(integration), medium-, and high-copy-number plasmid strains. At 18 h, the final time point,

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HB101-Ure-integration (lowest urease activity) produced large polycrystals. However, the size

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and abundance of polycrystals were lower for HB101/pBR322-Ure and HB101/pRS426-Ure

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(Figures 4b). The polycrystals formed by HB101-Ure-integration were most similar in size and

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form to those formed by S. pasteurii, which also had a substantial amount of unconsolidated crystal;

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however, the amount of large polycrystals in HB101-Ure-integration was higher (Figure 4b). Less

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material appeared to be incorporated into the polycrystals for strains with greater urease activity.

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Though HB101-Ure-integration produced the greatest number of large polycrystals at the final

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time point, this strain was slower than higher copy number strains and required more time (8 h) to

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produce large polycrystal numbers compared with HB101/pBR322-Ure or HB101/pRS426-Ure

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(Figures S9 and S10).

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Urease activities, which were varied via copy number, also affected abundance and

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morphology of crystals precipitated on sand. HB101-Ure-integration (lowest urease activity)

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formed large (>100 micrometer) structures with clear cell castings (Figure 4b). HB101/pBR322-

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Ure strain (medium urease activity) had similar crystals to the HB101-Ure-integration strain,

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though crystals were somewhat smaller, less abundant, and exhibited fewer castings (Figure 4b).


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The strain HB101/pRS426-Ure had the highest urease activity but also produced the fewest and

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smallest crystals of any of the strains and had no evidence of cell casting (Figure 4b), while larger,

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more abundant crystals were seen with lower urease activity in both flask and sand cultures

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(Figures 4b and 4c). This pattern could be attributable to the large difference in urease activity

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between single- and high-copy-number plasmid strains (2.1 U/g DCW vs. 5.6 U/g DCW,

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respectively). A different pattern was seen with the pBU11 deletion variants (aside from

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HB101/pBU11-∆rhaTs), likely because the range between their urease activities was smaller (2.6

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U/g DCW to 3.9 U/g DCW) and/or due to the role of additional gene fragments influencing crystal

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nucleation and growth.

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In summary, we designed, constructed, and compared different urease operons and

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expression levels for CaCO3 production in engineered Escherichia coli strains. We quantified

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urease expression and calcium uptake as well as characterized CaCO3 crystal phase and

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morphology for each of the 13 engineered strains. When evaluating possible correlations between

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each metric for the pBU11 deletion variants, there was only a weak relationship between urease

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activity, calcium uptake, and polycrystal size, suggesting that genes surrounding the urease gene

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cluster have an effect on crystal size. When evaluating the strains with different urease expression

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levels, there was a negative relationship between urease activity and polycrystal size. Also, the

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HB101-Ure-integration strain produced the most similar crystals to those precipitated by the native

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producer, S. pasteurii. We demonstrate for the first time that the rational-design of E. coli

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facilitates the production of a range of CaCO3 crystal sizes and morphologies. Controlling crystal

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size and shape may allow improved rheology, density, and potentially mechanical properties in

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existing applications for MICP, such as soil stabilization and concrete remediation. Furthermore,

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our findings enable novel applications for biogenic CaCO3, where controlling crystal size and

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shape can facilitate design of advanced biomaterials.

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Methods

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Plasmid construction

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The plasmid pBU11 was constructed with the entire sequence of the urease gene cluster

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from S. pasteurii ATCC 11859, including a segment of the plasmid pBR322 sequence26 that we

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received from the Bang laboratory.18 We sequenced the whole fragment inserted in pBU11 and

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blasted the sequences in NCBI Blast. From this, we predicted there were still 4 genes (ygjF, rhaTs,



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rhaTi, and phoE) besides the urease gene cluster (Figure 2a). We deleted each of them to construct

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plasmids pBU11-∆ygjF, pBU11-∆RhaTs, pBU11-∆RhaTi, and pBU11-∆phoE and deleted all of

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them to isolate the urease gene cluster to construct plasmid pBU11-ure with the Q5 Site-Directed

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Mutagenesis Kit (NEB E0552S) (Table 1).

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For the urease gene cluster, which includes genes ureA, ureB, ureC, ureE, ureF, ureG,

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ureD, we used an operon calculator to design urease expression operons with two strong

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constitutive promoters, J23119 (http://partsregistry.org/Part:BBa_J23119) and ProD. Then, we

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inserted two terminators and a synthetic RBS sequence in front of each gene (Figure 3a). Three

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different operons were created using three different RBS sequences (~10,000 au, 5,000 au, and

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1,000 au of translation initiation rates). We divided each operon into three fragments Ure-1, Ure-

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2 and Ure-3 and ordered them from Integrated DNA Technologies, Inc. The fragments Ure-1, Ure-

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2 and Ure-3 were co-transformed into Saccharomyces cerevisiae strain BY4709 (ATCC #200872)

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with the linearized vector pRS426, which allowed the entire urease operon to be assembled into

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pRS426. The transformed colonies were randomly picked and cultured in SC-Ura media,

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miniprepped using Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research), and PCR amplified

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for identification of the urease operon with the primer pair Ure-ID_F and Ure-ID_R (Table S1).

296

We sequenced the plasmids with the correct length and confirmed that the sequences were correct;

297

these confirmed plasmids were then designated as pRS426-Ure-RBS1000, pRS426-Ure-RBS5000,

298

and pRS426-Ure-RBS10000, respectively (Table 1). The strain HB101/pRS426-Ure-N was

299

created by reconstructing the urease synthetic pathway to reflect the relative differences in the

300

RBS translation initiation rates between genes as seen in the natural RBS; the optimized synthetic

301

RBS had translation initiation rates of 5,000 au, 10,000 au, 5,000 au, 10,000 au, 1,000 au, 5,000

302

au, and 1,000 au for genes ureA, ureB, ureC, ureE, ureF, ureG, and ureD, respectively) (Figure

303

3a, Table S1).

304

For the integration strain, three plasmids (pX2-Cas9, pSIM5, and gRNA) were needed. The

305

pX2-Cas9 plasmid was constructed by amplifying the Cas9 open reading frame from genomic

306

DNA of Streptococcus pyogenes strain SF370 (ATCC #700294) and cloning into the broad host

307

range plasmid pBTBX-2.27 The plasmid pSIM5 was acquired from the Court lab.28 The plasmid

308

gRNA was purchased from Addgene (#44251), and different gRNA plasmids were cloned using

309

CPEC.29 The vector pSS9, containing 600 bp homology arms, and the corresponding gRNA

310

plasmid (SS9_RNA) for integration is available in Addgene (#71655 and #71656, respectively).


Page 10 of 22

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311


Homology arms were amplified from boiled cells of E. coli BW2311.24

312 313

Strain construction

314

S. pasteurii ATCC 11859 was purchased from the American Type Culture Collection

315

(Bethesda, MD) and maintained in ATCC 1832 medium (BPU). E. coli HB101 was used as the

316

host strain for the urease operon transformation and integration. The constructed plasmids

317

pRS426-Ure-RBS1000, pRS426-Ure-RBS5000, and pRS426-Ure-RBS10000 were transformed

318

into E. coli HB101 to generate the strains HB101/pRS426-Ure-RBS1000, HB101/pRS426-Ure-

319

RBS5000, and HB101/pRS426-Ure-RBS10000 (Table 2). Urease operon integration was prepared

320

by transforming gRNA plasmid (SS9_RNA) and the linear template urease operons with the

321

homology arms (SS9-J23119-thl-atoDA-adc-adh-SS9) into the E. coli HB101 strain carrying the

322

temperature sensitive pSIM5 plasmid (lambda RED and temperature inducible) and a pX2-cas9

323

plasmid (arabinose inducible). The plasmid pSIM5 was induced at 42 °C for 15 min followed by

324

chilling on ice for 15 min. The cells were washed 3 times with 20% of the initial culture volume

325

of ddH2O (i.e., 10 mL washes for 50 mL culture). Following electroporation at 1800 V, the cells

326

were recovered in SOB +0.4% arabinose to induce Cas9. The cells were recovered 2 h before

327

plating on LB +0.4% arabinose +30 μg/mL kanamycin +100 μg/mL carbenicillin plates. Then, the

328

plates were cultured at 37 °C overnight. Next, we performed colony PCR screening by primers

329

SS9_Ure_F and SS9_Ure_R to choose the right integration strains, which were designated as

330

HB101/pRS426-Ure-RBS1000,

331

RBS10000 (Table S1, 2). The yeast strain used in this study was Saccharomyces cerevisiae strain

332

BY4709 (ATCC #200872). Transformed yeast strains were grown in SC-Ura media at 30 °C for

333

2–4 days. Yeast transformation associated recombination (TAR) cloning was conducted as

334

previously described30 but using LiAc transformation31 instead of electroporation.

HB101/pRS426-Ure-RBS5000,

and

HB101/pRS426-Ure-

335 336

Culture conditions

337

Details of the growth conditions for S. pasteurii ATCC 11859 are described in a previous

338

study;32 in summary, the culture of S. pasteurii was grown overnight in BPU medium at 30 °C. All

339

E. coli strains were grown in LB broth at 37 °C. E. coli integration strains were maintained in LB

340

broth containing 50 µM NiCl2 for urease activity, and E. coli plasmid strains were maintained in

341

LB broth containing 50 µM NiCl2 for urease activity and ampicillin (100 g/mL) for maintenance



342

of the plasmid.

343 344

CaCO3 precipitation

345

CaCO3 precipitation experiments were prepared in a previously reported urea–CaCl2

346

medium in which nickel (5 µM), ampicillin (100 µg/mL), and Ca2+ (50 mM for flask and 25.2 mM

347

for sand) were supplemented for the recombinant E. coli strains. 6 The experiment was conducted

348

in both flasks and sand cultures. For the experiments in flasks, cells were inoculated into 25 ml of

349

urea–CaCl2 medium to a final concentration of 1×107 cells/mL. The experiment was carried out in

350

a shaker at 37 °C for E. coli strains and at 30°C for S. pasteurii. For the experiments in sand, we

351

used disposable plastic columns (16 mm inner diameter) with 3g autoclaved Ottawa sand (particle

352

size 30-40 mesh, VWR, BDH9274). The column was inoculated with 2 mL of cells at OD600=0.2.

353

Then the urea-CaCl2 medium was added. Then, the columns were kept at 37°C for E. coli strains

354

and at 30°C for S. pasteurii for one week.

355

All samples were prepared in triplicate. At each interval (0, 2, 4, 8, 18 h), the replicates

356

were analyzed for pH, urease activity, using the Urease Activity Assay Kit (Sigma-Aldrich,

357

MAK120), and soluble Ca2+ concentration, using the Calcium Colorimetric Assay kit (Sigma-

358

Aldrich, MAK022). Also, CaCO3 crystal precipitates were studied by Raman spectroscopy to

359

confirm CaCO3 production and to identify individual CaCO3 crystal polymorphs; scanning

360

electron microscopy (SEM) was used to visualize crystal morphology; and SEM energy-dispersive

361

x-ray spectroscopy (EDS) was used to evaluate elemental composition. First, flask-cultured

362

samples were centrifuged, and sand-cultured samples were taken from the top of the sand. Then,

363

each sample was washed twice with deionized water and then evaporated on carbon tape. Raman

364

spectra were generated using Renishaw inVia (785 nm excitation, 50x objective). The baseline for

365

each spectrum was subtracted using an 11th order polynomial fit (Renishaw WIRE, v4.4).

366

Polymorphs were identified by the presence of unique peak signatures: calcite (285, 712, 1086 cm-

367

1),

368

650-690, triplet peak 720-780, triplet peak 1074-1090 cm-1), amorphous calcium carbonate (broad

369

peak 100-300, doublet peak 660-760, 1080 cm-1).33 Following Raman spectroscopy, the same

370

samples were coated with a 5-10 nm platinum coating for imaging with SEM (Hitachi SU3500, 5

371

kV accelerating voltage, 50 spot size for flask-cultured samples; JEOL 6480, 10 kV accelerating

372

voltage, 60 spot size for sand-cultured samples), and several crystals were assessed with SEM-

aragonite (210, doublet peak 701-705, 1087 cm-1), vaterite (multiple peaks 100-350, triplet peak


Page 12 of 22

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373


EDS (JEOL 6480, 15 kV accelerating voltage, 60 spot size).

374 375

AUTHOR INFORMATION

376

Corresponding Author

377

*E-mail: [email protected].

378

Author Contributions

379

L.L. and R.T.G. conceived the idea. L.L., C.H., R.L., and R.T.G. designed the experiments. L.L.,

380

C.H., and R.L. performed the experiments. L.L., C.H., R.L., and S.M.C. contributed data analysis.

381

A.N., J.C., W.V.S., and M.H. contributed assistance in designs and intellectual input. L.L., C.H.,

382

R.L., R.T.G., and S.M.C. wrote the manuscript.

383

Notes

384

The authors declare no competing financial interest.

385 386

Acknowledgments

387

The project or effort depicted was or is sponsored by the Defense Advanced Research Projects

388

Agency (Agreement HR0011-17-2-0039). The content of the information does not necessarily

389

reflect the position or the policy of the Government, and no official endorsement should be inferred.

390 391

Supporting Information:

392

SI includes additional results for all engineered strains (Figures S1-S10 and Table S1)

393 394

References

395 396 397 398 399 400 401 402 403 404 405 406

(1) Omori, M., and Watabe, N. (1980) The Mechanisms of Biomineralization in Animals and Plants: Proceedings, Tokai University Press. (2) Kaplan, D. L. (1998) Introduction to Biopolymers from Renewable Resources. In Biopolymers from Renewable Resources (Kaplan, D. L., Ed.), pp 1–29, Springer Berlin Heidelberg, Berlin, Heidelberg. (3) Dhami, N. K., Reddy, M. S., and Mukherjee, A. (2014) Application of calcifying bacteria for remediation of stones and cultural heritages, Front. Microbiol. 5, 304. (4) Hamilton, W. A. (2003) Microbially influenced corrosion as a model system for the study of metal microbe interactions: a unifying electron transfer hypothesis, Biofouling 19, 65–76. (5) Phillips, A. J., Gerlach, R., Lauchnor, E., Mitchell, A. C., Cunningham, A. B., and Spangler, L. (2013) Engineered applications of ureolytic biomineralization: a review, Biofouling 29, 715–733.



407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451

(6) Stocks-Fischer, S., Galinat, J. K., and Bang, S. S. (1999) Microbiological precipitation of CaCO3, Soil Biol. Biochem. 31, 1563–1571. (7) Hammes, F., and Verstraete*, W. (2002) Key roles of pH and calcium metabolism in microbial carbonate precipitation, Rev. Environ. Sci. Technol. 1, 3–7. (8) Jargeat, P., Rekangalt, D., Verner, M.-C., Gay, G., Debaud, J.-C., Marmeisse, R., and Fraissinet-Tachet, L. (2003) Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum, Curr. Genet. 43, 199–205. (9) Phillips, A. J., Lauchnor, E., Eldring, J. J., Esposito, R., Mitchell, A. C., Gerlach, R., Cunningham, A. B., and Spangler, L. H. (2013) Potential CO2 leakage reduction through biofilm-induced calcium carbonate precipitation, Environ. Sci. Technol. 47, 142–149. (10)Cunningham, A. B., Gerlach, R., Spangler, L., Mitchell, A. C., Parks, S., and Phillips, A. (2011) Reducing the risk of well bore leakage of CO2 using engineered biomineralization barriers, Energy Procedia 4, 5178–5185. (11)Ebigbo, A., Phillips, A., Gerlach, R., Helmig, R., Cunningham, A. B., Class, H., and Spangler, L. H. (2012) Darcy-scale modeling of microbially induced carbonate mineral precipitation in sand columns, Water Resour. Res., Wiley Online Library 48. (12)Dhami, N. K., Reddy, M. S., and Mukherjee, A. (2013) Biomineralization of calcium carbonates and their engineered applications: a review, Front. Microbiol. 4, 314. (13)Mobley, H. L. T., Garner, R. M., and Bauerfeind, P. (1995) Helicobacter pylori nickeltransport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions, Mol. Microbiol., Wiley Online Library 16, 97–109. (14)Mobley, H. L., Island, M. D., and Hausinger, R. P. (1995) Molecular biology of microbial ureases, Microbiol. Rev. 59, 451–480. (15)Ciurli, S., Marzadori, C., Benini, S., Deiana, S., and Gessa, C. (1996) Urease from the soil bacterium Bacillus pasteurii: Immobilization on Ca-polygalacturonate, Soil Biol. Biochem. 28, 811–817. (16)Mitchell, A. C., and Ferris, F. G. (2006) The Influence of Bacillus pasteurii on the Nucleation and Growth of Calcium Carbonate, Geomicrobiol. J., Taylor & Francis 23, 213– 226. (17)Mitchell, A. C., Phillips, A. J., Kaszuba, J., Hollis, W., Cunningham, A. B., and Gerlach, R. (2008) Microbially enhanced carbonate mineralization and the geologic containment of CO2, Geochim. Cosmochim. Acta 72, A636. (18)Bachmeier, K. L., Williams, A. E., Warmington, J. R., and Bang, S. S. (2002) Urease activity in microbiologically-induced calcite precipitation, J. Biotechnol. 93, 171–181. (19)Tate, C. G., Muiry, J. A., and Henderson, P. J. (1992) Mapping, cloning, expression, and sequencing of the rhaT gene, which encodes a novel L-rhamnose-H+ transport protein in Salmonella typhimurium and Escherichia coli, J. Biol. Chem. 267, 6923–6932. (20)Egan, S. M., and Schleif, R. F. (1993) A regulatory cascade in the induction of rhaBAD, J. Mol. Biol. 234, 87–98. (21)Grote, A., Hiller, K., Scheer, M., Münch, R., Nörtemann, B., Hempel, D. C., and Jahn, D. (2005) JCat: a novel tool to adapt codon usage of a target gene to its potential expression host, Nucleic Acids Res. 33, W526–31. (22)Gustafsson, C., Govindarajan, S., and Minshull, J. (2004) Codon bias and heterologous protein expression, Trends Biotechnol. 22, 346–353.


Page 14 of 22

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452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481


(23)Kosuri, S., Goodman, D. B., Cambray, G., Mutalik, V. K., Gao, Y., Arkin, A. P., Endy, D., and Church, G. M. (2013) Composability of regulatory sequences controlling transcription and translation in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A., National Academy of Sciences 110, 14024–14029. (24)Bassalo, M. C., Garst, A. D., Halweg-Edwards, A. L., Grau, W. C., Domaille, D. W., Mutalik, V. K., Arkin, A. P., and Gill, R. T. (2016) Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli, ACS Synth. Biol. 5, 561–568. (25)Tian, T., and Salis, H. M. (2015) A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons, Nucleic Acids Res. 43, 7137– 7151. (26)Lee, E. T., and Kim, S.D. (Yeungnam University, Gyongsan (Korea Republic). Department of Applied Microbiology). (oct1992) Purification and enzymatic characteristics of the Bacillus pasteurii urease expressed in Escherichia coli, Korean Journal of Applied Microbiology and Biotechnology (Korea Republic) 20. (27)Prior, J. E., Lynch, M. D., and Gill, R. T. (2010) Broad-host-range vectors for protein expression across gram negative hosts, Biotechnol. Bioeng. 106, 326–332. (28)Datta, S., Costantino, N., and Court, D. L. (2006) A set of recombineering plasmids for gram-negative bacteria, Gene 379, 109–115. (29)Quan, J., and Tian, J. (2011) Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries, Nat. Protoc. 6, 242–251. (30)Shao, Z., Zhao, H., and Zhao, H. (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways, Nucleic Acids Res. 37, e16. (31)Gietz, R. D., and Woods, R. A. (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method, Methods Mol. Biol. 313, 107–120. (32)Bang, S. S., Galinat, J. K., and Ramakrishnan, V. (2001) Calcite precipitation induced by polyurethane-immobilized Bacillus pasteurii, Enzyme Microb. Technol. 28, 404–409. (33)Wehrmeister, U., Soldati, A. L., Jacob, D. E., Häger, T., and Hofmeister, W. (2010) Raman spectroscopy of synthetic, geological and biological vaterite: a Raman spectroscopic study, J. Raman Spectrosc., Wiley Online Library 41, 193–201.

482



483

Page 16 of 22

Table 1. Plasmids used in this study Name

Description

Source

pRS426

Multiple copy plasmid with URA3 marker

ATCC

pSIM5

The temperature sensitive plasmid containing pSC101 origin

20

pX2-Cas9

Inducibly expressing Cas9

This study

gRNA

Directing Cas9 to introduce a precise double strand break (DSB) in the genome

Addgene

pSS9

Containing 600 bp homology arms to SS9 for one-step markerless genome integration in E. coli

Addgene

SS9_RNA

SS9-targeting gRNA for co-transformation with pSS9-IPA

Addgene

pRS426-UreRBS1000

pRS426-J23119-RBS1000-ureA-RBS1000-ureB-RBS1000-ureC-proDRBS1000-ureE-RBS1000-ureF-RBS1000-ureG-RBS1000-ureD

This study

pRS426-UreRBS5000


This study

pRS426-UreRBS10000


This study

pRS426- Ure_N


This study

pBR322-Ure

pBR322 with urease gene cluster from S. pasteurii ATCC 11859

This study

pRS426-Ure

pRS426 with urease gene cluster from S. pasteurii ATCC 11859

This study

pBU11

pBR322 with the entire sequence of the urease gene cluster from S. pasteurii ATCC 11859

(23)

pBU11-∆yjgF

pBU11 with yjgF deletion

This study

pBU11-∆rhaTs

pBU11 with rhaTs deletion

This study

pBU11-∆rhaTi

pBU11 with rhaTi deletion

This study

pBU11-∆phoE

pBU11 with phoE deletion

This study

pBU11-yjgF-ure

pBU11 with rhaTs, rhaTi, and phoE deletion

This study

484 485


Page 17 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

486


Table 2. Strains used in this study Name

Description

Source

MATalpha ura3delta0

ATCC

S. pasteurii

ATCC

E. coli HB101

F–, thi-1, hsdS20 (rB–, mB–), supE44, recA13, ara14, leuB6, proA2, lacY1, galK2, rpsL20 (strr), xyl5, mtl-1.

Promega

HB101/pBU11

E. coli HB101/pBU11

This study

HB101/pRS426-Ure-RBS1000

E. coli HB101/pRS426-Ure-RBS1000

This study



This study



This study

HB101/pRS426-Ure_N

E.coli HB101/pRS426-Ure_N

This study

HB101/pRS426-Ure

E. coli HB101/pRS426-Ure

This study

HB101/pBR322-Ure

E. coli HB101/pBR322-Ure

This study

HB101/pBU11-∆yjgF

E. coli HB101/pBU11-∆yjgF

This study

HB101/pBU11-∆rhaTs

E. coli HB101/pBU11-∆rhaTs

This study

HB101/pBU11-∆rhaTi

E. coli HB101/pBU11-∆rhaTi

This study

HB101/pBU11-∆phoE

E. coli HB101/pBU11-∆phoE

This study

HB101/pBU11-yjgF-ure

E. coli HB101/ pBU11-yjgF-ure

This study

Saccharomyces BY4709 Sporosarcina 11859

cerevisiae

pasteurii

HB101-Ure-integration

ATCC

E. coli HB101, urease gene cluster from S. pasteurii ATCC 11859

487 488


This study


489

a

b

c d

e f

490 491 492 493 494 495 496 497 498

Figure 1. Urease expression and calcite production with engineered E. coli by pathway optimization. (a) Schematic representation of calcite precipitation mechanism in engineered E. coli (modified from 12). (b) Urease activities of wild type S. pasteurii ATCC 11859, wild type E. coli HB101, and engineered HB101/pBU11. (c) Calcium uptake rates via Ca2+ concentrations over time of wild type S. pasteurii ATCC 11859, wild type E. coli HB101, and engineered HB101/pBU11. (d) The entire gene fragment from the pBU11 plasmid. Note: The genes in the green parts are predicted from protein sequence alignments. (e) Urease activities test in all the pBU11 plasmid deletion strains. (f) Ca2+ concentration over time for all deletion strains.


Page 18 of 22

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499 500 501 502 503 504


Figure 2. SEM images for S. pasteurii ATCC 11859 and engineered E. coli strains from flask cultures. (a) The relationship between urease activity and calcium uptake rate. (b) Group A: SEM images of CaCO3 produced by E. coli strains with lower urease activities (range = 2.6-3.3 U/g DCW). Group B: SEM images of CaCO3 produced by E. coli strains with higher urease activities (range = 3.9-4.8 U/g DCW).

505



a

RBS1 RBS1

RBS2

RBS2 RBS3

ureA ureB

yjgF

c

RBS3 RBS4

ureC

RBS4 RBS5

ureE

RBS5 RBS6

ureF

Urease gene cluster

b

RBS6 RBS7

ureG

ureD

rhaTs

rhaTl

pohE

d

506 507 508 509 510 511

Figure 3. The construction and verification of urease expression operons in E. coli. (a) The different strain construction strategies (i.e., synthetic RBS). (b) Urease activity and (c) calcium concentration profiles. (d) The translation initiation rates of different genes in the original and rationally-designed urease gene cluster.

512 513 514


Page 20 of 22

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a a

515

b

Urease activity

Crystal size

Urease activity

Crystal size

b

c c 516 517 518 519 520 521 522

HB101/pRS426-Ure

HB101/pBR322-Ure

HB101-Ure-integration

Figure 4. Calcite crystals are larger with lower urease expression for engineered E. coli strains. (a) The relationship between urease activity and calcium uptake rate. (b) SEM images of CaCO3 produced in flask cultures from engineered E. coli strains demonstrate that larger crystals HB101/pRS426-Ure HB101/pBR322-Ure HB101-Ure-integration are produced by strains with lower urease activity. (c) CaCO3 deposited on sand for the different copy-number strains.

523 524 525



526 527 528

Rational Control of Calcium Carbonate Precipitation by

529

Engineered Escherichia coli

530

Liya Liang1, Chelsea Heveran2, Rongming Liu1, Ryan T. Gill1,4, Aparna Nagarajan1, Jeffrey

531

Cameron1,3,5, Mija Hubler,2 Wil V. Srubar III,2 Sherri M. Cook*,2

532 533

1Renewable

and Sustainable Energy Institute (RASEI), 2Department of Civil, Environmental, and Architectural

534

Engineering, 3Department of Chemistry and Biochemistry, 4Department of Chemical and Biological Engineering,

535

University of Colorado Boulder, Boulder, CO 80303, United States, 5National Renewable Energy Laboratory,

536

Golden CO 80401, United States

537

538 539


Page 22 of 22

Rational Control of Calcium Carbonate Precipitation by Engineered

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