Konstruktie van plasmiden met genen van het tryptofaan operon van Escherichia coli K12 als genetische kenmerken

Chapter 1CONSTRUCTION OF NEW CLONING VEHICLES WITH GENES OF THETRYPTOPHAN OPERON OF Escherichia coli AS GENETIC MARKERSIn vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes Aos I, Ava I, Bgl I, Bgl II, Hind III, Hpa I, Pvu II, Sal I, Sst I and Xho I.The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trp E and trp A genes and contains single Bgl II and Sst I sites in trp E, a single Hind III site located between trp E and trp A, and single Eco RI, Sal I and Xho I sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trp B and trp A genes and single Bgl II, Hind III Eco RI, . Sal I and Xho I sites. Plasmids pHP39, (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one Eco RI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.CHAPTER 2THE CONSTRUCTION OF NEW VECTORS FOR THE CLONING OF PROMOTORSIn this paper we describe the construction and properties of vectors for the cloning of promotor-DNA fragments. The plasmids are derived from pBR313 (Bolivar et al., 1977), have several unique restriction sites and carry the trpA gene from Escherichiacoli as a genetic marker for the selection of promotor-DNA fragments. The selection is based on an enhancement of the growth rate of bacteria, due to an increase of the expression of trpA by the cloned promotor.The expression of trpA by a cloned promotor can be determined quantitatively, independently of the copy number of the vector. Determination of the trpA expression levels yields the apparent strength of the promotor, since the DNA segment located before trpA contains translational stop signals in the three reading frames.CHAPTER 3THE CONSTRUCTION OF NEW VEHICLES FOR THE CLONING OF TRANSCRIPTIONTERMINATION SIGNALSWe have constructed new plasmids that can be used to clone transcription terminator containing DNA fragments between the first gene of the tryptophan ( trp ) operon and the tetracycline resistance ( tet ) gene. Both genes are under control of the trp promotor. Therefore the presence of a transcription termination signal on cloned fragments can be monitored by a decrease in expression of the tet gene. The plasmids contain cloning sites at different distances from the translation start signal. Consequently a cloned DNA fragment can be translated in the three possible reading frames, offering the opportunity to distinguish terminators from translation polarity (pseudo-terminators). The usefulness of the plasmids was shown by the cloning of the trp terminator and of a pseudoterminator located in the trpB gene.CHAPTER 4The importance of the -35 and -10 region for the functioning of the promotor of the tetracycline resistance geneBy in vitro recombination we have joined the -35 region of the tet promotor to different sequences. This results in the formation of promotors with different -10 regions but with the same -35 region. The in vivo activity of these promotors was determined by measuring the expression of a downstream gene, trpA or trpE . The in vitro properties of the promotors were determined by measuring the protection of restriction sites within the promotor region by RNA polymerase. Our results suggest that the -35 region of the tet promotor is sufficient for the formation of a stable promotor-RNA polymerase complex at this region, whereas the formation of the initiation complex is determined by the structure of the -10 region.