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2.5 Tet array and pLau53
Fluorescent repressor-operator systems were first developed to visualise specific
DNA sites in eukaryotes ( Robinett et al. , 1996 ) but have been successfully used
to study chromosome segregation in E . coli , B . subtilis and Caulobacter crescentus
( Gordon et al. , 1997; Webb et al. , 1998; Viollier et al. , 2004 ). The system has since
been developed further to include dual expression of fluorescent proteins ( Lau et al. ,
2003 ) as well as creating road-blocks for the study of DNA-replication fork stalling
( Possoz et al. , 2006 ). These systems contain tandem copies of lacO and/or tetO
arrays inserted into specific chromosome regions. Originally, 256 copies were
inserted into the chromosome although currently as few as 64 have been used suc-
cessfully ( Fekete and Chattoraj, 2005 ). A plasmid (pLau53) producing C-terminal
CFP and YFP fusions of LacI and TetR, respectively, under the control of the arab-
inose promoter from pBAD has been constructed. The repressors bind specifically to
their cognate operators and facilitate visualisation of the loci (at least two) by fluo-
rescence microscopy. This system also allows for time-lapse analysis to visualise
chromosome dynamics in living cells.
3 FUNCTIONAL ANALYSIS
Once a gene fusion has been created, it is critically important to assess the function-
ality of the product. In cases in which the target gene is essential for growth, this can
be as simple as determining the growth kinetics with respect to wild-type strains (e.g.
Davies et al. , 2005 ). For non-essential genes, it is important to investigate the con-
ditions that would allow an assessment of the functionality. For example, a gene
essential for arabinose assimilation should be assessed for growth on arabinose as
the sole carbon source. Genes involved in stress responses should likewise be
assessed under the conditions that would allow the functionality to be determined.
In the case of transcription factors, there are numerous lacZ fusions available to
ensure that wild-type gene regulation is occurring in the recombinant strain. Finally,
cell morphology should be assessed under the microscope to ensure that the size and
shape of the cells are indistinguishable from the wild-type strain. A recent article
illustrating aggregation artefacts that can arise from fluorescent protein fusions
illustrates the importance of thoroughly investigating the functionality of the fusion
product to ensure costly and time-consuming experiments are not performed on
non-functional fusions ( Landgraf et al. , 2012 ).
4 GROWTH CONDITIONS
There are a variety of both liquid and solid growth media available to researchers.
Generally speaking, the richer the medium, the larger the cells and therefore more
spatial segregation within the cell. This is certainly the case when studying
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