Protein misfolding can occur as a result of several factors. It can be
due to overproduction of the protein in the cell, in which case the cell
lacks resources such as chaperones to fold the protein fast enough. This
can cause the proteins to misfold and form aggregate bodies. Proteins can
also misfold due to mutations that occur in the coding region of the
protein that can alter the amino acid sequence and thereby interrupting
the native structure of the protein, causing it to misfold and be
dysfunctional. Proteins can also misfold due to cellular stress such as a
change in pH, temperature and change in media. Due to lack of optimal
folding conditions proteins can form aggregate bodies and cause
activation of heat shock systems, chaperone systems and proteolytic
pathways which are involved in either refolding the proteins into their
native form or degrading the aggregate bodies. Proteins can also misfold
due to lack of localization. For example: if a periplasmic protein lacked
a signal sequence it will misfold in the cytoplasm because the conditions
are different in the two cellular compartments.
There are several heat shock pathways in E. coli which are actively
transcribed in response to cellular stress. This class of proteins are
called small heat shock proteins (sHsps). sHsps consist of proteins such
as ibpA, ibpB, DnaK, DnaJ, GroEL and GroES. Amongst these, IbpA and ibpB
are two different proteins that are activated as a result of cytoplasmic
stress response. IbpA and ibpB proteins are chaperones that are
responsible for refolding aggregated bodies and inclusion bodies into
their native conformation.
In our cytoplasmic stress detector circuit, we decided to fuse two
different promoter regions from two heat shock proteins, which are ibpAB
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly
higher output as a result of heat shock compared to the promoters
individually.
B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)
|
This fusion promoter will be connected to the registry part I13504 which
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence
of aggregation in the cell. We will be using MalE31 with a signal sequence
deletion (MalE31∆SS) which was designed by Betton et al. The native
E. coli protein MalE generally exported into the periplasmic space but
this mutated protein does not get exported to the periplasmic space due to
the signal sequence deletion. Also Betton et al designed MalE31such that
there are two amino acid changes in the protein and it misfolds. The
MalE31∆SS protein coding region will be used in order to induce
cytoplasmic protein stress in E. coli.
Ideally, this misfolded
MalE31∆SS should activate the plasmid system containing
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that
their protein is not being expressed in the cell because it is misfolding
and as a result getting degraded. Our circuit should also be activated
much faster than the native stress system because the ibpAB/fxsA promoter
is much more sensitive to the presence of aggregate bodies in the cell.
The promoter also gives a much higher output compared to the promoters
individually, which is the case in the E. coli genome which should allow
us to detect the fluorescence level much faster.
|
|