From Genes to Genomes by Dale Jeremy W.; von Schantz Malcolm; Plant Nicholas

Author:Dale, Jeremy W.; von Schantz, Malcolm; Plant, Nicholas
Language: eng
Format: epub
ISBN: 822573
Publisher: Wiley
Published: 2011-09-03T16:00:00+00:00

However, we have to remember that we are using a multi-copy vector. E. coli produces enough of the repressor protein to switch off the single copy of the promoter that it has in the chromosome, but this is not enough to switch off several hundred copies of this promoter. We refer to the repressor being titrated out by the presence of so many copies of the operator. So we have also to increase production of the repressor protein. The gene that codes for the repressor (the lacI gene) is not actually part of the lac operon; it has its own promoter. So one way of increasing production of the LacI repressor protein is by using a mutated version of the lacI gene that has a more active promoter (an up-promoter mutant). This altered lacI gene is known as lacIq. Or we can put the lacI gene onto the plasmid itself, so subjecting it to the same gene dosage effect and therefore increasing the production of LacI. Commonly, we would do both – i.e., put a lacIq gene onto the plasmid.

Adding IPTG to a laboratory culture is fine, but on a commercial scale it is still not an ideal solution. Adding IPTG to an industrial scale fermenter would be very expensive. An example of an alternative strategy is to use a promoter such as the trp promoter, which controls transcription of the tryptophan operon. This is subject to repression by tryptophan; E. coli switches off expression of the tryptophan operon when the enzymes encoded by it are not needed, i.e., if there is a plentiful supply of tryptophan. It is possible to monitor and control the availability of tryptophan so that there is an adequate supply during the growth phase, and then limit the supply of tryptophan when expression is required. You may be puzzled by this. If we stop supplying tryptophan, how is the cell going to make the protein that we want, which will probably contain some tryptophan residues? However, it is possible to supply a low level of tryptophan which is not enough to switch off the trp promoter, but will still enable production of the required protein. And we can then feed the culture continuously with a low level of tryptophan so that protein production will continue.

This may solve the problem in part, by removing the selective pressure imposed by excessive product formation. But there is still some element of selection imposed by the presence of so many copies of the plasmid, which may also slow growth rates. We can counter this by using a plasmid with a different replication origin, so that replication is tightly controlled at only one or two copies per cell (see Chapter 2). However, the level of expression achieved with a low-copy vector will be less than that achievable with a multi-copy plasmid, other things being equal. We can adopt a similar strategy to that described above for programming gene expression, using a so-called runaway plasmid. If the control of plasmid copy

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