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This is one of many Vector Coding Examples. Make sure you push Vector right to the back of the charger. Soluble expression and purification of hepatitis B core antigen HBcAg subgenotype B3 in Escherichia coli using thioredoxin fusion tag.
Escherichia coli thiol reductase has been utilized by Novagen to create an expression system for insoluble and poorly soluble proteins which normally form aggregates when overexpressed and become … Expand.
Highly Influential. View 4 excerpts, references methods and background. The Journal of Biological Chemistry. Expression of functional scorpion neurotoxin Lqq-V in E. Applied and Environmental Microbiology. It has been observed that fragments cut by NdeI type II restriction enzyme have low ligation efficiency. In ligation … Expand.
View 2 excerpts, references background and methods. Construction of a pET32a expression vector lacking the TrxA fusion protein. Author information Article notes Copyright and License information Disclaimer. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC. Abstract Background: Staphylococcus aureus is one of the most common causes of nosocomial infections and its resistance to antibiotics is a global concern. Objectives: In this study, high level of recombinant mature lysostaphin in Escherichia coli was produced by using pET32a expression vector. Materials and Methods: The S.
Results: PCR and sequencing results confirmed the successful cloning of the target gene into the vector. Conclusions: Our data showed that the recombinant mature lysostaphin protein produced by pET32a vector in E. Background Staphylococcus aureus is a major cause of both nosocomial and community-acquired infections worldwide that causes a wide range of diseases including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food-poisoning, carbuncles, and boils 1.
Objectives In the present study, we described a new expression system for producing r-lysostaphin in E. Materials and Methods 3.
Results 4. Expression and Purification of Recombinant Mature Lysostaphin The positive recombinant plasmid was transformed into the host, E. Open in a separate window. Figure 1. Discussion In this study, the mature lysostaphin recombinant protein from S.
Acknowledgments This study was conducted with financial assistance from Arak University of medical sciences, Iran, and we are grateful to for their invaluable contribution to this study. References 1. Kumar JK. Lysostaphin: an antistaphylococcal agent. Appl Microbiol Biotechnol. MRSA--time for a more pragmatic approach? J Hosp Infect. Emergence of vancomycin resistance in Staphylococcus aureus.
Glycopeptide-Intermediate Staphylococcus aureus Working Group. N Engl J Med. Staphylococcus, Micrococcus, and other catalase-positive cocci. Manual of clinical microbiology. Coagulase-negative staphylococcal infections. Infect Dis Clin North Am. The molecular organization of the lysostaphin gene and its sequences repeated in tandem. Mol Gen Genet. Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli.
Protein Expr Purif. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system. Appl Environ Microbiol. Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin. Microb Cell Fact. Plasmid DNA was prepared according to standard mini-preparation of plasmid Method.
Briefly, bacterial pellet was obtained from 1. The quality and quantity of purified plasmid DNA were assayed by 0. Primers were designed according to the full-length of lysostaphin gene sequence Gene Bank accession no: X Gene amplification of lysostaphin gene was performed in a total volume containing 20 ng of template DNA, 0.
The PCR products were analyzed in 0. The E. Then colonies were further analyzed by restriction enzyme digestion and PCR. The lysostaphin gene of the recombinant plasmid was sequenced by Sanger method. The expression host E. A single colony of transformed E. The culture was grown in an ODnm of 0. The plasmid DNA of S. The sequencing result was confirmed by comparing to database using basic local alignment search tool BLAST software. Enzyme digestion procedure, PCR assay, and sequencing result showed that target gene was inserted correctly into the recombinant plasmid pET-lys data are not show.
The positive recombinant plasmid was transformed into the host, E. The addition of IPTG induced the overexpression of approximately 42 kDa molecular weight recombinant protein.
The expressed protein was purified successfully via affinity chromatography using Ni-NTA resin Figure 1. The purification and dialysis process resulted in the yield of about 30 mg of purified protein from 1 L of E. A high-range molecular weight Marker is shown on left lane M. In this study, the mature lysostaphin recombinant protein from S. The obtained results showed that pET 32a system was very efficient.
Unlike an antibiotic, which interferes with bacterial growth, lysostaphin is highly effective in lysing S. Earlier methods for production of lysostaphin endopeptidase aimed to purify it from crude extract of S. In addition, mature lysostaphin is cleaved off the propeptide again using S. However, purification of wild-type lysostaphin is very difficult. Although several methods of lysostaphin production have reported, the yield and purity were very limited 11 , 17 , There are a number of reports for expression of lysostaphin endopeptidase in E.
Proendopeptidase was also expressed in eukaryotic system under the transcriptional control of Cytomegalovirus CMV promoter 9. The expression of recombinant prolysostaphin in Bacillus subtilis and B. Numerous proteins have been expressed in E. In several study, r-lysostaphin were produced through different pET vectors including pET28a with the yield of 22 mg, pET 23b with the yield of 20 mg, and pET15b with the yield of 11 mg of purified protein from 1 L of E.
A r-lysostaphin expressed in E. Further evaluation of the anti-Staphylococcal potential of lysostaphin as a therapeutic agent and its use as a laboratory reagent depends on the availability of large amounts of highly purified protein from a safe and nonpathogenic source.
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