Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression in Prokaryotic Systems
Unidirectional Tandem Gene Arrangement

Typically, the level of Gene Expression is proportional to the number of copies of the transcribed gene within host Cells. Consequently, increasing the plasmid copy number should theoretically boost the yield of the product encoded by the inserted gene. However, besides the cloned gene, a plasmid also harbors other transcribed sequences, such as Antibiotic Resistance genes. As its copy number rises, The Cell's energy resources become increasingly diverted toward producing plasmid-encoded Proteins, leading to a decline in the host cell's overall metabolic activity. One potential workaround is to insert multiple copies of the gene of interest into a low-copy-number plasmid. Yet, this introduces a technical challenge: arranging the genes so that all of them are correctly transcribed and translated. Simple end-to-end ligation results in random gene orientation, meaning that some genes will be expressed while others, oriented in the reverse direction, will not (Fig. 6.11).

To overcome this problem, one can use the restriction enzyme AvaI, which recognizes the sequence CTCGGG and cleaves DNA at the 5' end of the T residue. The Procedure is as follows: A plasmid containing this sequence is digested with AvaI, and the resulting sticky ends are filled in using DNA polymerase I. Next, an EcoRI linker (GAATTC) is attached to both blunt ends, and the plasmid is recircularized. The resulting plasmid contains a DNA segment with two AvaI sites flanking and overlapping the EcoRI site (Fig. 6.12, A and B)—namely, the sequence CTCGGGAATTCTCGGG (where the underlined bases represent the AvaI recognition sites). The target gene, complete with translational start and stop signals, is inserted into the EcoRI site and then excised from the plasmid using AvaI (Fig. 6.12, C). Such fragments feature non-identical sticky ends and therefore ligate in a single, uniform orientation during subsequent joining steps. A set of such unidirectional tandem gene copies can then be incorporated into an expression vector. Notably, the tandem array can insert in either of two orientations relative to the promoter, meaning that successful expression will occur in only 50% of the constructs.

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Fig. 6.11. Generation of randomly oriented tandem repeats. A. Cloned genes are excised from the cloning vector using the restriction endonuclease AbcI and separated from the vector DNA. B. Conditions are established to promote the ligation of the excised genes. Because The nucleotide sequences of both protruding ends of the genes are identical, the fragments can join in either orientation, resulting in tandem repeats composed of randomly oriented sequences.

Another approach relies on synthetic directional adapters—short oligodeoxynucleotides attached to the ends of linearized plasmid DNA and DNA fragments bearing the cloned gene. Upon ligation, these fragments assemble exclusively in a single orientation. This procedure is technically much simpler than the one employing the restriction endonuclease AvaI; furthermore, it does not require the target gene to be devoid of AvaI and EcoRI sites.

Experiments have already demonstrated that the expression level of interferon genes does indeed increase in proportion to the number of tandem gene copies, at least up to four copies per plasmid. Nevertheless, tandem repeats can occasionally prove unstable, and over time, some or even all of them may be lost by the plasmid.