An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.

Geotechnical observations of construction OF A large shallow SCL tunnel in soft ground

The Fort Canning Tunnel is the first road tunnel in Singapore to be built using the sprayed concrete lining (SCL) method. The major technical challenge of this was to construct a 15m wide tunnel by mining in soft ground under a shallow overburden of 3m to 9m. This paper describes the geotechnical investigations and monitoring controls for the safe and progressive execution of the works, such as soil investigations, trial forepoling works, surface settlement monitoring, tunnel settlement monitoring, face movement monitoring, and the observational approach to construction. The monitored field data showed the volume loss to range from 0.4% to 2.1%, and the observed surface settlement trough was found to agree well with the theoretical Gaussian trough. Other observations made include substantial surface settlements induced by the stress relief at and ahead of the tunnel face in spite of the forepoling umbrella, and the higher volume losses associated with higher overburden. Tunnel face movements were observed during installation of forepoling. These observations are of interest to engineers planning future SCL tunnels in similar conditions.