In the yearly Internationally Genetically Engineered Machines (iGEM) competition, teams of Bachelor's and Master's students design and build an engineered biological system using DNA technologies. Advising an iGEM team poses unique challenges due to the inherent difficulties of mounting and completing a new biological project from scratch over the course of a single academic year; the challenges in obtaining financial and structural resources for a project that will likely not be fully realized; and conflicts between educational and competition-based goals. This article shares tips and best practices for iGEM team advisors, from two team advisors with very different experiences with the iGEM competition.@en

Biofilm formation on membrane surfaces causes many operational problems such as a decrease in permeate flux and an increase in hydraulic resistance. In this study, the ability of bacteria to pass through microfiltration (MF) membranes and the growth potential of microfilterable bacteria were investigated in order to understand biofouling in MF-reverse osmosis (RO) integrated membrane systems. Growth of microfilterable bacteria in MF permeate was observed, indicating that not all MF membranes can guarantee the total rejection of bacteria. Changes in natural organic matter (NOM) characteristics and growth potential of bacteria during the treatment process are important factors in the occurrence of biofilm development in water treatment systems. Analysis of protein-like and humic-like substances in NOM of two successive RO stages revealed an increase in the concentrations of both biopolymers and humic substances of RO concentrates. Unexpectedly, the use of antiscalants was seen to enhance the growth of bacteria in the RO feed water in this study. Bacterial 16s rRNA pyrosequencing revealed that passing source water through the MF membranes dramatically changed bacterial community structure. The bacterial communities that passed through the MF steps primarily belonged to the family Comamonadaceae. However, several bacteria groups including Flavobacteriaceae, Sphingobacteriaceae and Sphingomonadaceae selectively composed the biofilm community formed on the RO membranes. Thus, understanding the selectivity and filterability of MF towards microorganisms involved in biofouling on RO membrane surfaces is crucial for the improvement of membrane-related operational processes.@en

Sustainable and personally tailored materials production is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different molecules in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials production techniques. In this paper, we describe the first steps toward the 3-dimensional printing of bacterial cultures for materials production and patterning. This methodology combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a commercial 3D printer was modified for bacterial systems, and new alginate-based bioink chemistry was developed. Printing temperature, printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resolution of printed structures. Our combination of 3D printing technology with biological systems enables a sustainable approach for the production of numerous new materials.@en

Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome. Results and discussion: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check. Conclusion: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.@en

Biofilms can grow on virtually any surface available, with impacts ranging from endangering the lives of patients to degrading unwanted water contaminants. Biofilm research is challenging due to the high degree of biofilm heterogeneity. A method for the production of standardized, reproducible, and patterned biofilm-inspired materials could be a boon for biofilm research and allow for completely new engineering applications. Here, we present such a method, combining 3D printing with genetic engineering. We prototyped a low-cost 3D printer that prints bioink, a suspension of bacteria in a solution of alginate that solidifies on a calcium-containing substrate. We 3D-printed Escherichia coli in different shapes and in discrete layers, after which the cells survived in the printing matrix for at least 1 week. When printed bacteria were induced to form curli fibers, the major proteinaceous extracellular component of E. coli biofilms, they remained adherent to the printing substrate and stably spatially patterned even after treatment with a matrix-dissolving agent, indicating that a biofilm-mimicking structure had formed. This work is the first demonstration of patterned, biofilm-inspired living materials that are produced by genetic control over curli formation in combination with spatial control by 3D printing. These materials could be used as living, functional materials in applications such as water filtration, metal ion sequestration, or civil engineering, and potentially as standardizable models for certain curli-containing biofilms.@en

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.@en

The influences of natural organic matter (NOM) and bacteriological characteristics on the biological stability of water were investigated in a full-scale drinking water treatment plant. We found that prechlorination decreased the hydrophobicity of the organic matter and significantly increased the high-molecular-weight (MW) dissolved organic matter, such as biopolymers and humic substances. High-MW organic matter and structurally complex compounds are known to be relatively slowly biodegradable; however, because of the prechlorination step, the indigenous bacteria could readily utilise these fractions as assimilable organic carbon. Sequential coagulation and sedimentation resulted in the substantial removal of biopolymer (74%), humic substance (33%), bacterial cells (79%), and assimilable organic carbon (67%). Rapid sand and granular activated carbon filtration induced an increase in the low-nucleic-acid content bacteria; however, these bacteria were biologically less active in relation to enzymatic activity and ATP. The granular activated carbon step was essential to securing biological stability (the ability to prevent bacterial growth) by removing the residual assimilable organic carbon that had formed during the ozone treatment. The growth potential of Escherichia coli and indigenous bacteria were found to differ in respect to NOM characteristics. In comparison with E. coli, the indigenous bacteria utilised a broader range of NOM as a carbon source. Principal component analysis demonstrated that the measured biological stability of water could differ, depending on the NOM characteristics, as well as on the bacterial inoculum selected for the analysis.@en

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.@en

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.@en

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.@en

In-situ resource utilization (ISRU) is increasingly acknowledged as an essential requirement for the construction of sustainable extra-terrestrial colonies. Even with decreasing launch costs, the ultimate goal of establishing colonies must be the usage of resources found at the destination of interest. Typical approaches towards ISRU are often constrained by the mass and energy requirements of transporting processing machineries, such as rovers and massive reactors, and the vast amount of consumables needed. Application of self-reproducing bacteria for the extraction of resources is a promising approach to reduce these pitfalls. In this work, the bacterium Shewanella oneidensis was used to reduce three different types of Lunar and Martian regolith simulants, allowing for the magnetic extraction of iron-rich materials. The combination of bacterial treatment and magnetic extraction resulted in a 5.8-times higher quantity of iron and 43.6% higher iron concentration compared to solely magnetic extraction. The materials were 3D printed into cylinders and the mechanical properties were tested, resulting in a 400% improvement in compressive strength in the bacterially treated samples. This work demonstrates a proof of concept for the on-demand production of construction and replacement parts in space exploration.@en

Microorganisms have developed an elaborate spectrum of mechanisms to respond and adapt to environmental stress conditions. Among these is the expression of dps, coding for the DNA-binding protein from starved cells. Dps becomes the dominant nucleoid- organizing protein in stationary-phase Escherichia coli cells and is required for robust survival under stress conditions, including carbon or nitrogen starvation, oxidative stress, metal exposure, and irradiation. To study the complex regulation of Dps in E. coli, we utilized time-lapse fluorescence microscopy imaging to examine the kinetics, input encoding, and variability of the Dps response in single cells. In the presence of an oxidative stressor, we observed a single pulse of activation of Dps production. Increased concentrations of H2O2 led to increased intensity and duration of the pulse. While lower concentrations of H2O2 robustly activated the Dps response with little effect on the growth rate, higher concentrations of H2O2 resulted in dramatically lower and highly varied growth rates. A comparison of cells exposed to the same concentration of H2O2 revealed that increased levels of Dps expression did not confer a growth advantage, indicating that recovery from stress may rely primarily upon variation in the amount of damage caused to individual cells.@en

Nacre (mother of pearl) is an attractive model for the development of new materials. Its sheet structure of alternating layers of calcium carbonate and an organic matrix confers it highly desirable properties such as high toughness and strength. In this study, we produce a nacre-inspired composite material using only bacterially-produced components. Calcium carbonate is crystallized via the action of ureolytic bacteria. After each crystallization event, we apply bacterially produced γ-polyglutamate (PGA) to the sample, which promotes layering compared to the PGA-free control. We show that the combination of these two compounds yields a layered material reminiscent of nacre, showing a way towards the biotechnological production of new, nacre-inspired materials.@en

Bacteria deficient in the DNA-binding protein from starved cells (Dps) are viable under controlled conditions but show dramatically increased mortality rates when exposed to any of a wide range of stresses, including starvation, oxidative stress, metal toxicity, or thermal stress. It remains unclear whether the protective action of Dps against specific stresses derives from its DNAbinding activity, which may exclude destructive agents from the chromosomal region, or its ferroxidase activity, which neutralizes and sequesters potentially damaging chemical species. To resolve this question, we have identified the critical residues of Escherichia coli Dps that bind to DNA and modulate iron oxidation. We uncoupled the biochemical activities of Dps, creating Dps variants and mutant E. coli strains that are defective in either DNA-binding or ferroxidase activity. Quantification of the contribution of each activity to the protection of DNA integrity and cellular viability revealed that both activities of Dps are required in order to counteract many differing stresses. These findings demonstrate that Dps plays a multipurpose role in stress protection via its dual activities, explaining how Dps can be of vital importance to bacterial viability over a wide range of stresses. @en