This chapter describes an Agrobacterium tumefaciens-mediated transformation protocol for torenia, a plant that has several useful characteristics and is primarily used for ornamental and experimental purposes. Leaf segments of torenia were co-cultured with A. tumefaciens containing a vector plasmid for 7 days at 22°C under dark conditions on Murashige and Skoog (MS) medium containing 1 mg/L benzyladenine, 1 mg/L indoleacetic acid, and 100 μM acetosyringone. Subsequent culturing at 25°C under a 16-h photoperiod with fluorescent light on MS medium containing 1 mg/L benzyladenine, 300 mg/L carbenicillin, and selection agent (300 mg/L kanamycin or 20 mg/L hygromycin) allowed for transformant selection. Transgenic shoots were obtained from green compact calli after 2-3 months of culture in the selection medium. This method can achieve a transformation rate of approximately 5%(transformants/explant).

We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using TcR as a marker. The transductant can then be freed of the TcR if required.

A family of cloning vectors derived from plasmid pACYC184 and, therefore, compatible with pBR322 and its derivatives (especially the pUC family of vectors), is described. They all contain a multiple cloning site (MCS) and the lacZ alpha reporter gene for easy cloning. They have been grouped in three sets:(i) six of the vectors contain a chloramphenicol-resistance (CmR)-encoding gene and each a different MCS with 16 unique restriction sites overall;(ii) another six vectors contain a kanamycin-resistance (KmR)-encoding gene and the same six MCS; and (iii) two CmR vectors that contain the SP6 and T7 promoters flanking the MCS and lacZ alpha reporter gene of pUC18/19.

Described here is a pair of small multi-copy kanamycin-resistance plasmids, containing the pUC lacZ alpha-complementation peptide and the pUC18 and pUC19 multiple cloning site. These plasmids and their derivatives allow simple and rapid transfer of inserts from one replicon to another without the necessity of purifying the insert from vector.

A lacZ gene without a promoter, but containing its ribosome-binding site, was cloned next to the kanamycin-resistance (KmR) gene of plasmid pUC4K, yielding a lacZ-KmR cassette. From the resulting plasmid, pKOK5, the lacZ-KmR cassette was recloned by means of BamHI into plasmid pKOK4, a mobilizable derivative of pBR322 which mediates ampicillin, chloramphenicol and tetracycline resistance. The lacZ-KmR cassette can be excised from pKOK5 or pKOK6 by digestion with BamHI, SalI or PstI. It can be used for insertion mutagenesis by ligation of the cassette to target DNA that has been linearized by one of these enzymes. Insertions can be selected by the KmR phenotype and mapped by digestion, e.g., with PstI and SalI. The orientation of the inserted cassette can be determined by digestion, e.g., with EcoRI or HindIII. Within the lacZ-KmR cassette, the transcription of the lacZ and the KmR genes are directed towards each other, and the two genes are separated by the bidirectionally active terminator from phage fd. In Escherichia coli, no transcription emanating from the cassette was detected. Transcription within DNA mutagenized by the cassette can be monitored by the promoterless lacZ gene. The lacZ-KmR cassette is currently used by us for the site-directed mutagenesis of hydrogen uptake gene-specific DNA from Rhizobium leguminosarum B10.

We have developed new Campylobacter shuttle vectors which are 6.5-6.8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site (MCS), the lacZ alpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene (KmR or CmR) from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a KmR marker and 17 unique cloning sites in two different orientations in lacZ alpha, allowing easy blue/white color selection. Plasmids pRY111 and pRY112 contain a CmR gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CmR gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.

A protocol for transformation of intact Enterococcus faecalis cells by electroporation was developed through a systematic examination of the effects of changes in various parameters, including (i) growth conditions;(ii) composition of the electroporation solution;(iii) electroporation conditions, such as field strength and resistance;(iv) size, concentration, and purity of DNA used for transformation; and (v) conditions used to select for transformants. Key features of this protocol include the use of exponential-phase cells grown in inhibitory concentrations of glycine and the use of an acidic sucrose electroporation solution. Frequencies of greater than 2 x 10(5) transformants per microgram of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 10(3) to 10(4) transformants per microgram of plasmid DNA with the same protocol. A novel Escherichia coli-Streptococcus and Enterococcus shuttle cloning vector, pDL276, was constructed for use in conjunction with the electroporation system. This vector features a multiple cloning site region flanked by E. coli transcription termination sequences, a relatively small size (less than 7 kb), and a kanamycin resistance determinant expressed in both gram-positive and gram-negative hosts. Various enterococcal and streptococcal DNA sequences were cloned in E. coli (including sequences that could not be cloned on other vectors) and were returned to the original host by electroporation. The vector and electroporation system was also used to clone directly into E. faecalis.

Cytosines in single-stranded DNA deaminate to uracils at 140 times the rate for cytosines in double-stranded DNA. If resulting uracils are not replaced with cytosine, C to T mutations occur. These facts suggest that cellular processes such as transcription that create single-stranded DNA should promote C to T mutations. We tested this hypothesis with the Escherichia coli tac promoter and found that induction of transcription causes approximately 4-fold increase in the frequency of C to U or 5-methylcytosine to T deaminations in the nontranscribed strand. Excess mutations caused by C to U deaminations were reduced, but not eliminated, by uracil-DNA glycosylase. Similarly, mutations caused by 5-methylcytosine to T deaminations were only partially reduced by the very short-patch repair process in E.coli. These effects are unlikely to be caused by differential repair of the two strands, and our results suggest that all actively transcribed genes in E. coli should acquire more C to T mutations in the nontranscribed strand.

Seven new streptococcal integration shuttle vectors have been constructed which contain different antibiotic-resistance-encoding genes capable of expression in both Streptococcus sp. and Escherichia coli. These plasmids can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The size, antibiotic resistance, and number of unique restriction sites available for cloning for each plasmid are as follows: pSF141 (7.6 kb, CmR and KmR, 7 sites), pSF143 (5.7 kb, TcR, 6 sites), pSF148 (7.3 kb, CmR and SpR, 7 sites), pDL285 (3.4 kb, KmR, 3 sites), pDL286 (3.1 kb, SpR, 4 sites), pSF151 (3.5 kb, KmR, 10 sites), pSF152 (3.2 kb, SpR, 9 sites). If these plasmids carry a fragment of streptococcal DNA they can specifically integrate into the chromosome via Campbell-like, homologous recombination. Therefore, they should be useful for gene inactivation, cloning, chromosomal walking, or linkage analysis in streptococci. The availability of these integration plasmids resistant to different antibiotics, along with the previously described plasmid, pVA891 (ErR), should also allow the construction of mutants possessing multiple insertionally inactivated genes useful for a variety of genetic studies.