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Revision as of 07:28, 10 October 2016
Description
Proof of concept
We have successfully proved our concept of plasmid-sensing logically
adjustable cell killer
(P-SLACKiller) at every step of our model and wet experiment.
See our final results here!
Problem we aim to solve
For plasmid-based expression system, the
plasmid segregational instability
has been the limit step in both scientific research
and large-scale industry production. Generation of plasmid-free
cells will sharply decrease the efficiency and
profit due to the slacker’s increasing proliferation ability
while producing no target substances.
Enhancing the plasmid stability is our main goal.
To control the intracellular plasmid numbers,
the first step is to sense the plasmid numbers.
Project overview
Equip the bacteria with a
plasmid-sensing logically adjustable cell killer (P-SLACKiller),
we select the inhibitor
protein as a signal which can indicate the intracellular plasmid numbers.
There is a basic rule: when the plasmid numbers are above the threshold,
we regard it as a normally-working bacterium and the P-SLACKiller won’t
start; however, when the plasmid numbers are
below the threshold, we judge it as a slacker and the P-SLACKiller
will kill these slackers.
Previous work done by others [reference]
Previous work realize the plasmid maintenance
through making the plasmid encode essential factors for the host.
Resistance screening and auxotrophic bacteria are dependent on the selecting process [1].
The plasmid-free bacteria cannot survive under specific selections, such as antibiotics.
According to the natural mechanisms for plasmid maintenance,
many new strategies are developed, so called
plasmid addiction system
(PAS) or post-segregational
killing system (PSK)
[2]. However, these mechanism are still under exploration.
Our special design
As we know, the employed antibiotics
must be removed
in pharmaceutical or GMP-based
fermentation processes and it’s not an
applicable option in industrial fermentation.
In our project, we aim to use an intracellular signal correlated with
the plasmid numbers and realize the selection process without any human interference.
This design will greatly simplify the spawn rejuvenation.
With no antibiotics added, we also want to improve the previous
approaches by realizing the quantification of plasmid copy numbers.
See our design.
Reference:
[1] Kroll J, Klinter S, Schneider C, et al. Plasmid addiction systems: perspectives and
applications in biotechnology. [J]. Microbial Biotechnology, 2010, 3(6):634-57.
[2] Friehs K. Plasmid copy number and plasmid stability. [J]. Advances
in Biochemical Engineering/biotechnology, 2004, 86:47-82.
