Plasmid recombinant

It is necessary to introduce the GDH recombinant plasmid into a GOGAT-defident mutant strain of AS1 because ... 

E. coli strain BL21(DE3) (F ompT ksdSafra ms ) gal dcm) (Novagen, Madison, WI, USA) was used as a host for recombinant OPH expression. Recombinant plasmids pTOH and pEOH that contain OPH gene fused with hexa-histidine affinity tag under trc and T7 promoter, respectively, as control vectors and pTTOH and pETOH that contain OPH gene fused with Tat signal sequence and hexa-histidine affinity tag under trc and T7 promoter, respectively, were used (Fig. 1). 

Fig.l. Gene maps of recombinant plasmids pTOH, pTTOH, pEOH, and pETOH. Abbreviations Pnc, trc promoter Px7, T7 and lac hybrid promoter Tat, twin-arginme TorA signal sequence of TMAO reductase OPH, organophosphoms hydrolase gene Hisg, hexa-histidine affinity tag. 

Fig. 1 Schematic outline of procedures employed in the synthesis of a cDNA gene copy from a polyadenylated mRNA template, insertion of the cDNA into a bacterial plasmid vector by a homopolymer tailing strategy, and cloning of the recombinant plasmid in an Escherichia coli host.

Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 7 1513-1523. 

Once the recombinant vectors have been produced, they are used to transform host cells. In the example of the plasmid pBR322, the host cells are bacteria. Once transformed, the bacteria are plated on selective media so that bacteria transformed with a recombinant plasmid can be easily identified. In the case of plasmid pBR322 shown in Figure 1-6-3, bacteria with recombinant plasmids would be resistant to ampicillin but sensitive to tetracycline. 

Cloning procedure (in bacteria) Recombinant plasmids transfected into bacteria Antibiotic resistance used to select bacteria with recombinant plasmids Blot growth plate and probe with P-DNA for gene, or with Tantibody for protein... 

Chemical recombination of vector insertion into the host cell recombining plasmid genes into the host genetic complement Replication of host cell... 

Transform the reaction into E. coli cells and confirm recombination by digesting miniprep DNA with Xbal. Cre-recombined plasmids that lost the stop cassette produce two bands of 460 bp and 2.9 kb. Nonrecombined plasmids produce bands of 1.3 and 2.9 kb. After sequence confirmation using the M13 forward and reverse primers, prepare a Maxiprep from one confirmed clone. 

Colonies harboring the recombinant plasmid are confirmed by using PCR-based colony identification methods. 

The confirmed recombinant plasmid is sequenced in the connecting region or in all regions. 

Cells that grow on tetracycline but not on tetracycline + ampicillin contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates. 

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