STABILITY OF trp OPERON RECOMBINANT PLASMID IN E. coli

1981 ◽  
pp. 95-100 ◽  
Author(s):  
Tadayuki Imanaka ◽  
Hiroshi Tsunekawa ◽  
Shuichi Aiba
Keyword(s):  
Recombinant Plasmid ◽  
E Coli ◽  
Trp Operon

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

1982 ◽  
Vol 152 (1) ◽  
pp. 57-62
Author(s):  
M Blumenberg ◽  
C Yanofsky
Keyword(s):  
Regulatory Region ◽  
Enteric Bacteria ◽  
Evolutionary Divergence ◽  
Citrobacter Freundii ◽  
Initial Portion ◽  
Trp Repressor ◽  
E Coli ◽  
Weak Expression ◽  
Dyad Symmetry ◽  
Trp Operon

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.

scholarly journals Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
Wayan T. Artama ◽  
Yulia Sari ◽  
Didik Tulus Subekti ◽  
Soenarwan Hery Poerwanto ◽  
Jarot Subandono
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Messenger Rna ◽  
Secretory Protein ◽  
Gene Encoding ◽  
Isolation System ◽  
Complementary Dna ◽  
E Coli ◽  
Local Isolate ◽  
Cell Target

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2

scholarly journals Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto
Keyword(s):  
Protein Expression ◽  
Newcastle Disease ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Free Protein ◽  
F Protein ◽  
E Coli ◽  
Protein Expression System ◽  
Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

Recombinant plasmid mobilization betweenE.colistrains in seven sterile microcosms

1997 ◽  
Vol 43 (6) ◽  
pp. 534-540 ◽  
Author(s):  
P. Lebaron ◽  
P. Bauda ◽  
N. Frank ◽  
M. C. Lett ◽  
B. Roux ◽  
...  
Keyword(s):  
Recombinant Plasmid ◽  
Recombinant Dna ◽  
Transfer Functions ◽  
Genetically Engineered ◽  
Plasmid Transfer ◽  
Transfer Rates ◽  
E Coli ◽  
Attached Biomass ◽  
Plasmid Mobilization ◽  
Dna Plasmid

Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra−, mob−) from one E. coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments. These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut. GEMOs survived in the same way as the host strains in all microcosms. Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm. The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.Key words: recombinant DNA, plasmid transfer, mobilization, conjugation, microcosm.

scholarly journals Interaction of a colicinogenic factor with a resistance factor and with the fertility factor F inEscherichia coliK-12

1971 ◽  
Vol 17 (2) ◽  
pp. 151-159 ◽  
Author(s):  
Pearl Cooper
Keyword(s):  
Resistance Genes ◽  
Recombinant Plasmid ◽  
Resistance Factor ◽  
E Coli ◽  
Fertility Factor ◽  
Plasmid Incompatibility ◽  
R Factor ◽  
Transmissible Plasmid ◽  
K 12 ◽  
R Factors

SUMMARYThe resistance (R)-factor R538–1drdof three F-like R-factors tested can cause the transfer of the non-transmissible ColV determinant fromE. coliV. The col factor, once transferred toE. coliK-12 strains, was shown to be related to the fertility factor, F, on the basis of entry exclusion and plasmid incompatibility. The col factor was found to recombine with R538–1drd, yielding a transmissible plasmid comprised of the col determinant and the sex factor of the R-factor, but not the resistance genes. The recombinant plasmid was found to be incompatible with both R538–1drdand F, and excluded the entry of R538–1drdbut not F.

scholarly journals Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

2007 ◽  
Vol 51 (12) ◽  
pp. 4401-4409 ◽  
Author(s):  
Jun-ichi Wachino ◽  
Keigo Shibayama ◽  
Hiroshi Kurokawa ◽  
Kouji Kimura ◽  
Kunikazu Yamane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Recombinant Plasmid ◽  
Coli Strain ◽  
Methyltransferase Activity ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Rrna Molecule ◽  
A Site

ABSTRACT We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

scholarly journals VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

2005 ◽  
Vol 11 (1) ◽  
pp. 61-66
Author(s):  
Ira Djajanegara ◽  
Wayan Artama ◽  
Retno Lestari ◽  
Sabar Pambudi
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Restriction Site ◽  
Standard Procedure ◽  
Pcr Amplification ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Coding ◽  
Sds Page ◽  
Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

scholarly journals EKSPRESI GEN PENYANDI b-XILOSIDASE DALAM SISTEM pHIS1525/ Bacillus megaterium MS941

2008 ◽  
Vol 13 (2) ◽  
pp. 157-161
Author(s):  
Sri Sumarsih ◽  
Ni Nyoman Tri Puspaningsih ◽  
Sofijan Hadi ◽  
Ami Soewandi J.S.
Keyword(s):  
Bacillus Megaterium ◽  
Recombinant Plasmid ◽  
Culture Medium ◽  
Protein Band ◽  
Extracellular Protein ◽  
Protoplast Transformation ◽  
E Coli ◽  
Sds Page ◽  
Xylosidase Activity

The aim of this research was to express the β-xylosidase gene in the pHIS1525 or Bacillus megaterium MS941 system. The xyl gene was amplified from pTP510 and cloned into pHIS1525 in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant B. megaterium MS941 on solid LB medium containing tetracycline (10 μg/ml). The expression of β-xylosidase was assayed using 0.2 percent methylumbelliferyl-β-D-xyloside (MUX) and the proteins were analyzed by SDS-PAGE method. The b-xilosidase activity was determined toward p-nitrophenyl-b-Dxylopyranoside (pNPX) as a substrate and p-nitrofenol releasing was measured by UV/Vis spectrophotometer at λ 405 nm. This research showed that recombinant B. megaterium MS941 expressed the β-xylosidase gene (xyl) and secreted it into the culture medium. The SDS-PAGE analysis of extracellular protein (culture medium) showed a 60,0 kD protein band. The recombinant Bacillus megaterium MS941 expressed and secreted the β-xilosidase into culture medium 5 hours after adding 5 percent xylose. The b-xylosidase activity was 0.441 unit/ml toward pNPX as a substrate.

scholarly journals Clonning and expression of recombinant carbapenemase KPC-2 enzyme in E. coli cytoplasm

2016 ◽  
Vol 19 (2) ◽  
pp. 27-37
Author(s):  
Phuong Nhat Tran ◽  
Phuong Thi Kim Huynh ◽  
Trang Thi Phuong Tran ◽  
Thuoc Linh Tran ◽  
Van Hung Pham
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clinical Research ◽  
Affinity Chromatography ◽  
Recombinant Plasmid ◽  
Broth Culture ◽  
E Coli ◽  
Recombinant Vector ◽  
Carbapenem Resistant ◽  
Sds Page ◽  
Encoding Genes

Production of KPC-type carbapenemase is the most common carbapenem resistant mechanism in Klebsiella pneumoniae. The expression level of KPC in these strains is different and is mostly required other mechanisms to reach the higher resistant level such as porin lost or co-expression of extended spectrum β-lactamase (ESBL). To better understand the expression of KPC enzyme, the KPC-2 encoding genes from clinical isolated K. pneumoniae were cloned into pET28a plasmid. The recombinant plasmids containing of kpc-2 gene were subsequently transformed into E. coli OmniMax and were screened in kanamycine added LB media to select E. coli possessing of recombinant plasmid. Carbapenemase activity in the broth culture was checked in LB broth supplemented with 4 µg/mL of ertapenem and the expression induced with IPTG was checked by SDS-PAGE method. The results showed that this recombinant vector was capable of effective expression of KPC-2 protein in E. coli and this strain could be grown in LB broth supplemented with 4 µg/mL of ertapenem. A half of the target protein was soluble in the supernatant however it could be successfully collected from a HistrapHP affinity chromatography column. The result of this report is one of resources for further studies and applications of this KPC-2 protein in clinical research.

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