DNA Condensation by Peptide-Conjugated PAMAM Dendrimers. Influence of Peptide Charge.

Nucleic acid delivery to cells is an important therapeutic strategy that requires the transport of nucleic acids to intracellular compartments and their protection from enzymatic degradation. This can be achieved through the complexation of the nucleic acids with polycations. Poly(amidoamine) (PAMAM) dendrimers and peptide-conjugated dendrimers have been investigated as delivery vectors. Inspired by these studies and the role of flexible peptide domains in protein-DNA interactions, we studied the impact of conjugating two peptides (tails) to generation 2 (G2) PAMAM dendrimers on DNA condensation and polyplex formation. Using gel electrophoresis, dye exclusion assays, atomic force microscopy, and Monte Carlo simulations, it is shown that the steric impact of neutral peptide tails is to hinder the formation of DNA-G2 polyplexes composed of multiple DNA chains. If the tails are negatively charged, which results in overall neutral G2 conjugates, then the interaction of G2 with DNA is hindered. Increasing the net positive charge of the tails resulted in the complexation capacity of G2 with the DNA being restored. While DNA complexation is obtained for a similar net charge balance for G2 and G2 conjugates with positive tails, fewer of the latter are required to achieve a comparable condensation degree. Furthermore, it is shown that about 40% of the DNA remains accessible to binding by small molecules. Overall, this shows that tuning the net charge of peptide tails conjugated to PAMAM dendrimers offers a handle to control the complexation capacity of DNA, which can be explored as a novel route for optimization as gene delivery vehicles.

A universal approach to gene expression engineering.

In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5' regulatory sequences (ARES) consisting of promoters and 5' untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus and Streptomyces lividans, and the eukaryote yeast Saccharomyces cerevisiae.

mCherry contains a fluorescent protein isoform that interferes with its reporter function.

Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expression studies. In this study, we identified the short protein isoform, traced its origin, and determined the extent of the issue within the family of red fluorescent protein. Our analysis showed that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. We provided a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

Identification and Cross-Characterisation of Artificial Promoters and 5' Untranslated Regions in Vibrio natriegens.

Vibrio natriegens has recently gained attention as a novel fast-growing bacterium in synthetic biology applications. Currently, a limited set of genetic elements optimised for Escherichia coli are used in V. natriegens due to the lack of DNA parts characterised in this novel host. In this study, we report the identification and cross-characterisation of artificial promoters and 5' untranslated regions (artificial regulatory sequence, ARES) that lead to production of fluorescent proteins with a wide-range of expression levels. We identify and cross-characterise 52 constructs in V. natriegens and E. coli. Furthermore, we report the DNA sequence and motif analysis of the ARESs using various algorithms. With this study, we expand the pool of characterised genetic DNA parts that can be used for different biotechnological applications using V. natriegens as a host microorganism.

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution.

Water-in-oil emulsion droplets are an attractive format for ultrahigh-throughput screening in functional metagenomics and directed evolution applications that allow libraries with more than 107 members to be characterized in a day. Single library members are compartmentalized in droplets that are generated in microfluidic devices and tested for the presence of target biocatalysts. The target proteins can be produced intracellularly, for example, in bacterial hosts in-droplet cell lysis is therefore necessary to allow the enzymes to encounter the substrate to initiate an activity assay. Here, we present a titratable lysis-on-demand (LoD) system enabling the control of the cell lysis rate in Escherichia coli. We demonstrate that the rate of cell lysis can be controlled by adjusting the externally added inducer concentration. This LoD system is evaluated both at the population level (by optical density measurements) and at the single-cell level (on single-cell arrays and in alginate microbeads). Additionally, we validate the LoD system by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations in the µM range. The LoD system yields sufficient release of the intracellularly produced enzymes to bring about a detectable quantity of product (measured by fluorescence in flow cytometry of double emulsions), while leaving viable cells for the downstream recovery of the genetic material.

Importance of the 5' regulatory region to bacterial synthetic biology applications.

The field of synthetic biology is evolving at a fast pace. It is advancing beyond single-gene alterations in single hosts to the logical design of complex circuits and the development of integrated synthetic genomes. Recent breakthroughs in deep learning, which is increasingly used in de novo assembly of DNA components with predictable effects, are also aiding the discipline. Despite advances in computing, the field is still reliant on the availability of pre-characterized DNA parts, whether natural or synthetic, to regulate gene expression in bacteria and make valuable compounds. In this review, we discuss the different bacterial synthetic biology methodologies employed in the creation of 5' regulatory regions - promoters, untranslated regions and 5'-end of coding sequences. We summarize methodologies and discuss their significance for each of the functional DNA components, and highlight the key advances made in bacterial engineering by concentrating on their flaws and strengths. We end the review by outlining the issues that the discipline may face in the near future.

Dual UTR-A novel 5' untranslated region design for synthetic biology applications.

Bacterial 5' untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5' UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5' UTRs in a single expression cassette. The dual UTR consists of two 5' UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

Effect of design geometry, exposure energy, cytophilic molecules, cell type and load in fabrication of single-cell arrays using micro-contact printing.

In this study a range of factors influencing the fabrication of single-cell arrays (SCAs) are identified and investigated. Micro-contact printing was used to introduce spots coated with polyethyleneimine or Matrigel on glass surfaces pre-coated with polyethylene glycol. Unmodified E. coli, Synechococcus sp., Chlamydomonas reinhardtii as well as diverse mammalian cells including HUVEC, AAV293, U87, OHS, PC3, SW480, HepG2 and AY-27 were successfully immobilised onto the chemically coated spots. The developed SCAs show high cell viability and probability for capturing single-cells. A discrepancy between the size and shape of the squares described in the design file and the actual structures obtained in the final PDMS structure is characterised and quantified. The discrepancy is found to be depending on the exposure energy used in the photolithography process as well as the size of the squares and their separation distance as detailed in the design file. In addition to these factors, the effect of the cell density loaded onto the patterned surfaces is also characterised. The systematic characterisation of key parameters that need to be optimised prior to the fabrication of SCAs is essential in order to increase the efficiency and reproducibility of future fabrication of SCAs for single-cell studies.

Genome Mining of Streptomyces sp. YIM 130001 Isolated From Lichen Affords New Thiopeptide Antibiotic.

Streptomyces bacteria are recognized as an important source for antibiotics with broad applications in human medicine and animal health. Here, we report the isolation of a new lichen-associating Streptomyces sp. YIM 130001 from the tropical rainforest in Xishuangbanna (Yunnan, China), which displayed antibacterial activity against Bacillus subtilis. The draft genome sequence of this isolate strain revealed 18 putative biosynthetic gene clusters (BGCs) for secondary metabolites, which is an unusually low number compared to a typical streptomycete. Inactivation of a lantibiotic dehydrogenase-encoding gene from the BGC presumed to govern biosynthesis of a thiopeptide resulted in the loss of bioactivity. Using comparative HPLC analysis, two peaks in the chromatogram were identified in the extract from the wild-type strain, which were missing in the extract from the mutant. The compounds corresponding to the identified peaks were purified, and structure of one compound was elucidated using NMR. The compound, designated geninthiocin B, showed high similarity to several 35-membered macrocyclic thiopeptides geninthiocin, Val-geninthiocin and berninamycin A. Bioinformatics analysis of the geninthiocin B BGC revealed its close homology to that of berninamycins.

Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002.

BACKGROUND: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations. RESULTS: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly. CONCLUSIONS: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications.

In vitro studies of DNA condensation by bridging protein in a crowding environment.

The macromolecules of the bacterial cell occupy 20-40% of the total cytosol volume, and crowded environments have long been known to compact and stabilize DNA. Nevertheless, investigations on DNA-protein binding are generally performed in the absence of crowding, which may yield an incomplete understanding of how nucleoid-assembling proteins work. A family of such proteins, abundant in Gram-negative bacteria, is the histone-like nucleoid structuring proteins (H-NS). Herein, the synergistic role of macromolecular crowding (mimicked using polyethylene glycol, PEG) and H-NS was investigated using fluorescence correlation spectroscopy (FCS) and enzyme protection assays. We show that crowding enhances the binding of H-NS to the AT-rich tracks of the DNA, where it preferentially binds to, protecting these tracks towards enzyme digestion, inducing some DNA condensation, and inhibiting the biological function of DNA. We further suggest that the looping of DNA chains, induced by H-NS, contributes to the synergistic effect of DNA-binding protein and crowding agents, on DNA condensation.

Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.

BACKGROUND: Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. RESULTS: In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. CONCLUSIONS: Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven plasmids. However, there were differences at the pathway level as genes associated with degradation of phenolic compounds, nicotine, phenylalanine, styrene, ethylbenzene, and ethanolamine were detected only in the Psychrobacter strains reported in this study while they were absent among the other publicly available Psychrobacter genomes. In addition, six gene candidates were identified by genome mining and shown to possess T1, T2 and T3 copper binding sites as the main signature of the three-domain laccases. P11F6-LMCO and P11G5-LMCO2 were recombinantly expressed and shown to be active when ABTS and guaiacol were used as substrates.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.

A new and improved host-independent plasmid system for RK2-based conjugal transfer.

Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

BioBrick assembly standards and techniques and associated software tools.

The BioBrick idea was developed to introduce the engineering principles of abstraction and standardization into synthetic biology. BioBricks are DNA sequences that serve a defined biological function and can be readily assembled with any other BioBrick parts to create new BioBricks with novel properties. In order to achieve this, several assembly standards can be used. Which assembly standards a BioBrick is compatible with, depends on the prefix and suffix sequences surrounding the part. In this chapter, five of the most common assembly standards will be described, as well as some of the most used assembly techniques, cloning procedures, and a presentation of the available software tools that can be used for deciding on the best method for assembling of different BioBricks, and searching for BioBrick parts in the Registry of Standard Biological Parts database.

Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.

BACKGROUND: XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration. RESULTS: By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer. CONCLUSION: According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

Combinatorial engineering for heterologous gene expression.

Tools for strain engineering with predictable outcome are of crucial importance for the nascent field of synthetic biology. The success of combining different DNA biological parts is often restricted by poorly understood factors deriving from the complexity of the systems. We have previously identified variants for different regulatory elements of the expression cassette XylS/Pm. When such elements are combined they act in a manner consistent with their individual behavior, as long as they affect different functions, such as transcription and translation. Interestingly, sequence context does not seem to influence the final outcome significantly. Expression of reporter gene bla could be increased up to 75 times at the protein level by combining three variants in one cassette. For other tested reporter genes similar results were obtained, except that the stimulatory effect was quantitatively less. Combination of individually characterized DNA parts thus stands as suitable method to achieve a desired phenotype.

A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.

BACKGROUND: Production of recombinant proteins in bacteria for academic and commercial purposes is a well established field; however the outcomes of process developments for specific proteins are still often unpredictable. One reason is the limited understanding of the performance of expression cassettes relative to each other due to different genetic contexts. Here we report the results of a systematic study aiming at exclusively comparing commonly used regulator/promoter systems by standardizing the designs of the replicon backbones. RESULTS: The vectors used in this study are based on either the RK2- or the pMB1- origin of replication and contain the regulator/promoter regions of XylS/Pm (wild-type), XylS/Pm ML1-17 (a Pm variant), LacI/PT7lac, LacI/Ptrc and AraC/PBAD to control expression of different proteins with various origins. Generally and not unexpected high expression levels correlate with high replicon copy number and the LacI/PT7lac system generates more transcript than all the four other cassettes. However, this transcriptional feature does not always lead to a correspondingly more efficient protein production, particularly if protein functionality is considered. In most cases the XylS/Pm ML1-17 and LacI/PT7lac systems gave rise to the highest amounts of functional protein production, and the XylS/Pm ML1-17 is the most flexible in the sense that it does not require any specific features of the host. The AraC/PBAD system is very good with respect to tightness, and a commonly used bioinformatics prediction tool (RBS calculator) suggested that it has the most translation-efficient UTR. Expression was also studied by flow cytometry in individual cells, and the results indicate that cell to cell heterogeneity is very relevant for understanding protein production at the population level. CONCLUSIONS: The choice of expression system needs to be evaluated for each specific case, but we believe that the standardized vectors developed for this study can be used to more easily identify the nature of case-specific bottlenecks. By then taking into account the relevant characteristics of each expression cassette it will be easier to make the best choice with respect to the goal of achieving high levels of protein expression in functional or non-functional form.

Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette.

BACKGROUND: The XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Activation of transcription from the Pm promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements xylS (X), Pm (P) and the 5'-untranslated region (U) that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels. RESULTS: We combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene bla (resistance to penicillin). Combination of optimized elements stimulated bla expression up to 75-fold (X, P and U combined) relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, celB (encoding phosphoglucomutase) and the human growth factor gene gm-csf. Protein production from these genes is much more efficient than from bla in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for bla. We also integrated a single copy of the expression cassette with each gene into the E. coli chromosome and found that the expression level from this single copy was higher for bla than for the wild-type plasmid system, while it was lower for celB and gm-csf. CONCLUSION: Our results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in E. coli. For one reporter gene (bla) this allowed for more protein production from a single gene copy integrated on the chromosome, compared to the wild-type plasmid system. The approach described here should in principle be applicable for improvement of any expression cassette.