Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion

2019 ◽  
Vol 32 (10) ◽  
pp. 433-441
Author(s):  
Ichiro Matsumura ◽  
Donian Chyong
Keyword(s):  
Ribosomal Protein ◽  
Heterologous Gene Expression ◽  
Ribosomal Protein Gene ◽  
Host Cells ◽  
Recombinant Bacteria ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Novel Strategy ◽  
Statistical Noise

Abstract The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, ‘ribosome pegging’, to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.

Improvement in segregational stability of recombinant plasmids by retransformation of E. coli host cells

1993 ◽  
Vol 15 (8) ◽  
Author(s):  
Jorge Lavastida ◽  
Lilia Colon ◽  
Michael Ward ◽  
BenjaminC. Stark
Keyword(s):  
Host Cells ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Segregational Stability

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein

1990 ◽  
Vol 10 (12) ◽  
pp. 6544-6553
Author(s):  
J A All-Robyn ◽  
N Brown ◽  
E Otaka ◽  
S W Liebman
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Structural Genes ◽  
E Coli ◽  
Mouse Protein ◽  
Translational Ambiguity

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.

scholarly journals Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

1990 ◽  
Vol 10 (12) ◽  
pp. 6544-6553 ◽  
Author(s):  
J A All-Robyn ◽  
N Brown ◽  
E Otaka ◽  
S W Liebman
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Structural Genes ◽  
E Coli ◽  
Mouse Protein ◽  
Translational Ambiguity

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.

scholarly journals Regulation of Ribosomal Protein Synthesis in Mycobacteria: The Autogenous Control of rpsO

2021 ◽  
Vol 22 (18) ◽  
pp. 9679
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Oksana S. Bychenko ◽  
Irina V. Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Shuttle Vector ◽  
Ectopic Expression ◽  
Egfp Expression ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Ribosomal Protein Synthesis

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals An Assessment of the Viability of Lytic Phages and Their Potency against Multidrug Resistant Escherichia coli O177 Strains under Simulated Rumen Fermentation Conditions

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 265
Author(s):  
Peter Kotsoana Montso ◽  
Caven Mguvane Mnisi ◽  
Collins Njie Ateba ◽  
Victor Mlambo
Keyword(s):  
Escherichia Coli ◽  
Rumen Fluid ◽  
Multidrug Resistant ◽  
Rumen Fermentation ◽  
Ruminal Fermentation ◽  
Fermentation Conditions ◽  
E Coli ◽  
Cell Counts ◽  
Milk Contamination ◽  
Novel Strategy

Preslaughter starvation and subacute ruminal acidosis in cattle are known to promote ruminal proliferation of atypical enteropathogenic Escherichia coli strains, thereby increasing the risk of meat and milk contamination. Using bacteriophages (henceforth called phages) to control these strains in the rumen is a potentially novel strategy. Therefore, this study evaluated the viability of phages and their efficacy in reducing E. coli O177 cells in a simulated ruminal fermentation system. Fourteen phage treatments were allocated to anaerobic serum bottles containing a grass hay substrate, buffered (pH 6.6–6.8) bovine rumen fluid, and E. coli O177 cells. The serum bottles were then incubated at 39 °C for 48 h. Phage titres quadratically increased with incubation time. Phage-induced reduction of E. coli O177 cell counts reached maximum values of 61.02–62.74% and 62.35–66.92% for single phages and phage cocktails, respectively. The highest E. coli O177 cell count reduction occurred in samples treated with vB_EcoM_366B (62.31%), vB_EcoM_3A1 (62.74%), vB_EcoMC3 (66.67%), vB_EcoMC4 (66.92%), and vB_EcoMC6 (66.42%) phages. In conclusion, lytic phages effectively reduced E. coli O177 cells under artificial rumen fermentation conditions, thus could be used as a biocontrol strategy in live cattle to reduce meat and milk contamination in abattoirs and milking parlours, respectively.

scholarly journals Isolation and structural analysis of a ribosomal protein gene inD.melanogaster

1988 ◽  
Vol 16 (11) ◽  
pp. 4915-4926 ◽  
Author(s):  
F. Rafti ◽  
G. Gargiulo ◽  
A. Manzi ◽  
C. Malva ◽  
G. Grossi ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Ribosomal Protein ◽  
Protein Gene ◽  
Ribosomal Protein Gene

scholarly journals Isolation and characterisation of a murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene

1987 ◽  
Vol 15 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Pierre Belhumeur ◽  
Gary D. Paterno ◽  
Guy Boileau ◽  
Jean-Michel Claverie ◽  
Daniel Skup
Keyword(s):  
Ribosomal Protein ◽  
Cdna Clone ◽  
Protein Gene ◽  
Ribosomal Protein Gene
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