Herbert L. Heyneker

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.

Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This ‘hybrid’ gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable.

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12

A 2.3-kb PstI- ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.

CONSTRUCTION OF THREE HYBRID PROMOTERS AND THEIR PROPERTIES IN ESCHERICHIA COLI

SUMMARY This paper describes three hybrid promoters which are functional in Escherichia coli . In the case of the first hybrid promoter ( tac I) sequences upstream of position −20 were derived from the trp promoter and sequences downstream of position -20 were derived from the lac -UV5 promoter. This hybrid promoter is seven times stronger than the lac -UV5 promoter. It can be repressed by the lac-repressor and induced by isopropyl-β-D-thiogalactoside (IPTG). In the case of second hybrid promoter ( tac II), we used the DNA sequences upstream of the Hpa I site (which is located in the Pribnow box of the trp -promoter) and fused those sequences to a synthetic DNA fragment of 46 bp. The sequence of the synthetic fragment creates a new Pribnow-box which is followed by the lac -operator. Downstream from the lac -operator are nucleotides that code for a Shine-Dalgarno (SD) sequence. The Shine-Dalgarno sequence is flanked by two restriction sites which allows us to exchange different Shine-Dalgarno sequences. Thus, we constructed an inducible promoter with a portable Shine Dalgarno sequence which forms an active ribosome binding site when fused to the start codon of a foreign gene. The tac II promoter is as efficient as the tac I promoter. The third hybrid promoter (rac 5-16) is a hybrid between the rrnB promoter and the lac UV5 promoter. Its structure resembles that of the tac I promoter. At the junction, in the area of -20, three unique restriction sites were introduced. This makes it possible to change the distance and the nucleotide sequence between the –35 area and the −10 area (the Pribnow-box).

Synthesis, cloning, and expression of hormone genes in Escherichia coli.

Publisher Summary This chapter focuses on the synthesis, cloning, and expression of hormone genes in Escherichia coli. The genetically engineered bacteria is a valuable source of peptide hormones. The chapter also focuses on the insulin project and provides an overview of the recombinant DNA and chemical DNA synthesis techniques. Genes for large hormones, proteins, and enzymes can be made by chemical DNA synthesis. However, in most cases it is preferable to isolate the natural DNA sequence by cloning a reverse transcript of the appropriate messenger RNA (mRNA). This approach to gene isolation works well if a method is available for detecting the desired gene sequence among the shotgun library of bacterial or viral clones. Detection of the desired clone usually is difficult except for the most abundant protein, and thus mRNA species. The most important function for synthetic DNA can be in trimming, lengthening, or changing natural sequences to obtain efficient expression of the desired peptide product.