Enhance Plasmid Stability Research Articles

Titration of replication activity by increasing ARS dosage in yeast plasmids.

The rep1 region of the yeast mitochondrial genome, a putative replication origin, contains a weak autonomously replicating sequence (ARS). Nucleotide-sequence and deletion analyses have identified two 11-base pair ARS consensus sequences, numerous near matches to the ARS core, and a region of curvature that may contribute to ARS function. Based on the amplified nature of petite-derivative mitochondrial DNA encompassing this locus, we have constructed plasmids containing an increasing dosage of ARS elements. The rep1 ARS element can have an additive effect on plasmid stability when present either as a tandem dimer or as an unlinked pair. However, the presence of a third ARS copy does not further enhance plasmid stability. These results indicate that measurable dosage effects can be defined only in circumstances where weak ARS elements are employed, and that plasmid maintenance within yeast cells is saturable and varies among the different sequences promoting replication.

Effects of anaerobic conditions on biomass production and plasmid stability in an immobilized recombinantE. Coli

Cellular aggregation is a natural phenomenon shown by microorganisms which allows the cells to form biofilms. The study of immobilized microorganisms requires the development of microtechniques to evaluate their metabolism and stability. In aerobic conditions, the immobilization of recombinant microorganisms in carrageenan gel beads has been successfully explored to enhance plasmid stability when growth in the support was oxygen‐limited. A series of anaerobic continuous cultures of free and immobilized Escherichia coli B (pTG201) and E. coli HB 101 (pKBF 367–11) have been developed, without selection pressure. In continuous culture of free cells, both exhibited segregational instability. The existence of strong competition between cells containing plasmid (P+ cells) and cells devoid of plasmids (P— cells) was favourable to the colonization of the bioreactor by the faster growing P— cells. Furthermore, pTG 201 was mainly dimeric while the plasmid copy number of pKBF 367–11 increased from 80 to 270, until p...

Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine.

The Fab region of an IgG2b antibody (AM7B2.1) reactive to the herbicide atrazine was cloned into a plasmid vector using the polymerase chain reaction and two sets of degenerate oligonucleotide primers designed to mimic the amino acid variation at the N-termini of kappa L-chains and gamma H-chains. These primers also provide a secretion signal fused precisely to the antibody gene sequence for secretion of the mature antibody. A further set of universal oligonucleotide primers was developed for the direct sequencing of the VH and CH1 regions of gamma H-chains and the VL and CL regions of kappa L-chains without subcloning and were used to determine the sequence of this antibody. The kappa L-chain was found to not possess a conserved Cys residue at position 23 and the implications of this observation are discussed. The cloned genes were expressed in Escherichia coli using a commercially available T7 RNA polymerase-based plasmid. The clones were also expressed in a T7 RNA polymerase-based system containing an attenuated version of the T7 RNA polymerase promoter, plus a lac promoter placed in an antisense orientation, to enhance plasmid stability. The expressed products were confirmed as atrazine reactive by binding to an atrazine derivative conjugated with alkaline phosphatase.

Enhanced plasmid stability through post-segregational killing of plasmid-free cells

In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, β-galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10−11 with full promoter induction under non-selective conditions.

Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3‐dioxygenase activity in free and immobilized Escherichia coli cells

In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used.

Immobilization of recombinant Escherichia coli in silicone polymer beads

Porous silicone polymer beads containing viable colonies of Escherichia coli were formed using dimethylsiloxane prepolymer and silicone fluid in a gentle emulsion-polymerization procedure. Viability, cell outgrowth, and amylase production were examined in studies using both batch and continuous cultivation. Significant production of viable cells and amylase was observed. Enhanced plasmid stability through immobilization was demonstrated when cells were grown in M9/glycerol medium without antibiotic.

The use of the immobilization of whole living cells to increase stability of recombinant plasmids in escherichia coli

Immobilization of whole living cells was used as an experimental approach to enhance plasmid stability in cultured recombinant micro-organisms. pTG201 plasmid which is very unstable in continuous cultures with free cells, was found to be extremely stable in continuous cultures with immobilized cells. To elucidate the mechanism by which immobilization increases the plasmid stability, we analyzed the growth of pTG201-containing E. coli W3101 cells within the gel beads. We found that in immobilized continuous culture, plasmid-free segregants were not detected even after 240 generations. This appears to be due to the mechanical properties of the gel-bead system that allow only a limited number of cell divisions (10–16) to occur in each clone of cells before the clone escapes from the gel bead. This number of generations is not sufficient for the plasmid-free cells to appear within the cavities compared to what was observed in a free-system (plasmid-free segregants were detected after a lag period of approximately 25–30 generations). Even when they appear, they cannot overcome the culture. From the data described in this paper we conclude that cells released from the gel beads at any time during continuous culture are cells which are issued from cells grown in the cavities for only 10–16 generations.