Reporter-expressing recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) represents an excellent tool to understand the biology of and ease studying viral infections in vitro and ...in vivo. The broad range of applications of reporter-expressing recombinant viruses is due to the facilitated expression of fluorescence or bioluminescence readouts. In this chapter, we describe a detailed protocol on the generation of rSARS-CoV-2 expressing Venus, mCherry, and NLuc that represents a valid surrogate to track viral infections.
Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca
) signaling networks are implicated in most abiotic stress responses, ...heat-triggered changes in cytosolic Ca
were investigated in
leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca
reporter with an m
herry reference domain fused to an intensiometric Ca
reporter
CaMP6
. Relative changes in Ca
were estimated based on CGf's apparent
around 220 nM. The ratiometric output provided an opportunity to compare Ca
dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca
signals, comprised of three different signatures showing similarly rapid rates of Ca
influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca
signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth Ca
oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable Ca
(78%). Together, these contrasting examples of heat-triggered Ca
responses in leaves and pollen highlight the diversity of Ca
signals in plants, inviting speculations about their differing kinetic features and biological functions.
Background
Corynebacterium glutamicum is an important chassis for industrial applications. The low efficiency of commonly used genome editing methods for C. glutamicum limits the rapid multiple ...engineering of the bacterium.
Main Methods and Major Results
In this study, chromosome‐borne expression of cas9 and recET from Escherichia coli K12‐MG1655 was achieved to avoid toxicity to the strain, increase the probability of homologous recombination, and reduce loss of viability caused by double‐strand breaks. Constitutive strong promoters, such as P45, Ptrc, and PH36, were used to replace PglyA and to expand the application of the CRISPR‐Cas9 system. By using this system, a C. glutamicum strain producing L‐homoserine to 22.1 g per L in a 5‐L bioreactor after 96 h was obtained.
Conclusions and Implications
Through the application of visualized fluorescent protein, the process of plasmid curing was optimized, obtain a continuous and rapid CRISPR‐Cas9 genome editing system. The method described here could be useful to construct C. glutamicum mutant rapidly.
The metabolic engineering of C. glutamicum requires efficient genome editing tools. In this study, the authors optimized the previous CRISPR‐Cas9 system by promoter replacement, then modified the metabolic pathway of C. glutamicum to block the competitive pathways and enhance the biosynthetic pathways through this system, resulting in a strain with high L‐homoserine production. This work would greatly promote the rapid construction of the target C. glutamicum mutants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective ...wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both
and
, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (
ssp.
MG1363) carrying the pExu vector encoding the fluorescence protein mCherry
MG1363 (pExu:
) was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:
vector, was demonstrated
by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0-168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated
bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel.
Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For ...exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 μg L
of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common P
promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that P
failed to drive expression during the product synthesis phase but that use of two synthetic promoters (P
and P
) enabled strong constitutive expression during the entire process. Using P
, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L
in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3'-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L
nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Oxidation-reduction chemistry is fundamental to the metabolism of all living organisms, and hence quantifying the principal redox players is important for a comprehensive understanding of cell ...metabolism in normal and pathological states. In mammalian cells, this is accomplished by measuring oxygen partial pressure (pO2) in parallel with free and enzyme-bound reduced nicotinamide adenine dinucleotide (phosphate) H (NAD(P)H) and flavin adenine dinucleotide (FAD, a proxy for NAD+). Previous optical methods for these measurements had accompanying problems of cytotoxicity, slow speed, population averaging, and inability to measure all redox parameters simultaneously. Herein we present a Förster resonance energy transfer (FRET)-based oxygen sensor, Myoglobin-mCherry, compatible with fluorescence lifetime imaging (FLIM)-based measurement of nicotinamide coenzyme state. This offers a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular pO2 and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states.
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•A new method for concurrent oxygen and metabolic cofactor imaging.•This method quantifies the relationship between metabolism and changing oxygenation.•Optical and fluorescence lifetime redox ratios versus intracellular oxygenation can infer shifts in metabolic behavior.•Tandem oxygen and metabolic cofactor imaging shows intracellular heterogeneity.•Response to changing oxygenation and metabolism between cells and cell lines is heterogeneous.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Virally expressed fluorescent reporter mCherry fusion protein does not adequately demonstrate its presence in fixed tissue.•Immunohistochemical amplification of mCherry can rescue this deficit.
...Fluorescent reporter proteins are a powerful tool being increasingly integrated into biological experiments. Their utility spans techniques such as live-cell imaging, validating transgene expression, and studying cell-type specific anatomy. As these reporters become more widely used, it is necessary to fully understand their benefits and limitations. One such recently developed red fluorescent protein, mCherry, has been well utilized due to its stability, brightness, and pH resistance. In the course of an experiment using the fluorescent reporter protein mCherry fused to a G-protein coupled receptor (mCherry fusion protein), our lab discovered a notable inability for the fusion protein to faithfully produce fluorescent signal representative of its expression in fixed tissue. Here, we demonstrate the importance of immunohistochemical amplification in tissue injected with various adeno-associated viruses (AAVs), containing mCherry fusion protein as a reporter. Our findings demonstrate that antibody amplification consistently provides a stronger signal when mCherry fusion protein is used as a reporter protein.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for ...studying the
in vivo
physiological activities of LAB including localization, adhesion, and colonization.
Lactiplantibacillus plantarum
WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome
via
double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of
in vivo
WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the
bsh
gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB
in vivo
in order to determine the mechanism of its probiotic function.
Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and ...arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.
Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. ...This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2pRCR12 and M9MG6-B2pRCR12, expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2pRCR12 and M9MG6-B2pRCR12. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.
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