plasmid vector
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2022 ◽  
Author(s):  
Abhiney Jain ◽  
Anaísa Coelho ◽  
Joana Madjarov ◽  
Smilja Todorovic ◽  
Ricardo O. Louro ◽  
...  

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1.


Author(s):  
Shinto James ◽  
Vikas Jain

We introduce OLIVAR (Orientation seLection of Insert in Vector through Antisense Reporter) as a novel selection strategy for the insertion of protein-coding genes into vector backbones. As a proof-of-concept, we have engineered a plasmid vector, pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System), for gene cloning in E. coli. With pGRASS, positive clones can be effortlessly distinguished from negative clones after blunt-end cloning. The vector not only screens clones with an insert but also for its correct orientation. The design further allows for the expression of recombinant protein from the T7 promoter in an appropriate host bacterium. With this vector, we are able to reduce the entire cloning workflow into a single step involving a 2-h reaction at room temperature. We believe that our cloning-cum-screening system presented here is extremely cost-effective and straightforward and can be applied to other vector systems and domains such as phage display and library construction.


2021 ◽  
Vol 19 ◽  
Author(s):  
Wei Tan ◽  
Longjia Dong ◽  
Xuexing Shi ◽  
Qian Tang ◽  
Dianming Jiang

Objective: The aim of the study was to investigate the mechanism by which p75 neurotrophin receptor (p75NTR) affects mitochondrial damage and neuronal apoptosis in spinal cord injury (SCI). Methods: After the establishment of SCI rat models, short hairpin (sh) RNA of p75NTR and control sh-RNA were injected into SCI rats, respectively. On days 1, 7 and 21 after SCI, the severity of SCI and cell apoptosis in SCI rats were determined as well as the recovery of hind limb performance and p75NTR expression. After spinal cord neurons were transfected with p75NTR overexpression plasmid or empty plasmid vector or cotransfected with overexpression plasmids of p75NTR and neurotrophic tyrosine receptor kinase3 (NTRK3), the expression levels of p75NTR and NTRK3 were quantified. Moreover, we detected the apoptosis and proliferation rates of the neurons in addition to the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in the neurons. The binding between p75NTR and NTRK3 was confirmed via Co-immunoprecipitation (Co-IP). Results: The rat spinal cords in the Model group were notably damaged after SCI accompanied by increased apoptosis and decreased locomotor function. The expression of p75NTR was significantly upregulated after SCI. The aforementioned injuries were remarkably ameliorated in response to injection of sh-p75NTR. p75NTR overexpression induced mitochondrial damage and neuronal apoptosis in spinal cord neurons, while the promotive effects were perturbed by NTRK3 overexpression. Furthermore, p75NTR directly bound to and downregulated NTRK3. Conclusion: Both in vivo and in vitro experiments showed that p75NTR aggravates mitochondrial damage and neuronal apoptosis in SCI through downregulating NTRK3.


2021 ◽  
Author(s):  
Om P. Singh ◽  
Shobhna Mishra ◽  
Ankita Sindhania ◽  
Taranjeet Kaur ◽  
U. Sreehari ◽  
...  

AbstractBackgroundAnopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable based on the number of ridges present on the egg’s floats and the dimension of eggs. Recently, these forms have been designated as sibling species based on the fixed differences in the DNA sequence of the first intron of the odorant-binding protein-1 (AsteObp1). In this study, we evaluated the utility of this neutral marker in designating sibling species or identifying biological forms.MethodsField collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of floats on egg-ridge. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sanger’s method, either directly or after cloning with a plasmid vector.ResultsAsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 12 haplotypes exhibiting nucleotide-as well as length-polymorphism (90-to-121 bp). A majority of the field samples were heterozygous (up to 89% in the field populations). The phasing of haplotypes in heterozygotes through Sanger’s sequencing was challenging due to indels (1-to-24 bp) at multiple loci. No specific haplotype or monophyletic clade of intron-1 was found associated with a specific biological form. The inbreeding coefficient for this marker was close to zero in field and laboratory populations which refute the existence of sibling species based on the AsteObp1 marker.ConclusionsAsteObp1 cannot serve as a marker for the identification of biological forms of An. stephensi. The probable existence of sibling species in An. stephensi based on the AsteObp1 intron-1 is refuted.


2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 328-329
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma

Abstract Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2–3 mm2) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO2 incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with >80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.


Author(s):  
Teo, Y. L. ◽  
Toh, W. K. ◽  
Tor, X. Y. ◽  
Ho, C.-L. ◽  
Loh, P. C. ◽  
...  

2021 ◽  
pp. 107981
Author(s):  
Carly Boye ◽  
Sezgi Arpag-McIntosh ◽  
Michael Francis ◽  
Scott DeClemente ◽  
Aislin West ◽  
...  

2021 ◽  
Vol 189 ◽  
pp. 106323
Author(s):  
Muhammad Ehsaan ◽  
Jonathan Baker ◽  
Katalin Kovács ◽  
Naglis Malys ◽  
Nigel P. Minton

2021 ◽  
Author(s):  
Kaihang Wang ◽  
Lizhi Zhou ◽  
Tingting Chen ◽  
Qiong Li ◽  
Jiajia Li ◽  
...  

Abstract BackgroundThe various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli.ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci—lpxM, lpxP, lpxL, eptA, gutQ and kdsD­—were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. ConclusionReengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.


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