| dc.description.abstract | The main goal of this work is to map how bacterial nucleoid associated proteins (NAPs)
condense DNA in tandem with a crowded environment, since this may provide useful
information for research where DNA compaction and protein binding is of high importance,
such as protein expression, gene therapy and cancer research. The isolated effects
of NAPs on condensation are subject to constant investigation, but little is known about
the combination of both protein binding and crowding effects in vitro. The combined
effects of crowding and condensing agents on DNA compaction are investigated with fluorescence
spectroscopy (FS), fluorescence correlation spectroscopy (FCS), atomic force
microscopy (AFM), cell-free expression assays and electrophoresis mobility shift assays
(EMSAs). Plasmid pSB-M2g-1-17 was harvested from DH5-α Escherichia coli, and subjected
to polyethylene glycol (PEG) and the condensing polyamine spermine. Histone-like
nucleoid-structuring protein (H-NS) was over-expressed, purified and its activity confirmed. A 4017 bp amplicon from template plasmid pSB-E1g, including a T7 promoter and
a gene encoding green fluorescent protein (GFP), was used to quantify DNA compaction
and protein expression under influence of PEG and H-NS. The combined effect of PEG
and spermine was found to reduce the hydrodynamic volume of DNA to 5% of its initial
value, while spermine and PEG alone reduced it to 33% and 23%, respectively, although
with considerable standard deviations. 0.5 to 1.0 µM H-NS was found sufficient to inhibit
cell-free expression of GFP, and 5 µM induced visual DNA-aggregation by AFM. Novel
insight in synergism was attained, as crowding was shown to greatly reduce the amount of
H-NS required for condensation as measured by FCS. | |
