Plasmid Locking Assembly for Sustaining Multiple Insert DNA
Indian Institute of Technology – Madras, India
Abstract
In order to achieve locking and unlocking of gene function
in a combinatorial manner, a generic circuit was designed
(Fig 1.) based on the principle of plasmid instability using
which a gene function can be unlocked only after a predefined unique series of chemical/physical inputs. The
circuit was reduced to a 3-plasmid entity with the inputs as
antibiotic washes. As a proof of concept, constitutive RFP
and CFP expression cassettes were constructed in different
antibiotic backbones, and co-transformed into E.coli.
Growth of the differentially transformed strains, as well as
the plasmid loss, was measured against time, in various
media in order to prove the directionality of plasmid loss.
Fig 1: The gene of interest is "locked" or repressed by the inhibitor in the
plasmid 2. The plasmid linkage determines a unique order of directional loss,
by means of toxin expression otherwise. This unique, predetermined order of
growth conditions serves to express, or “unlock” the gene of interest.
Theory
Any
extra-chromosomal
genetic material introduced
into the cell tends to disappear
over the generations, unless it
confers a selective survival
advantage over the cells that do
not possess the plasmid.
Plasmids
which
confer
resistance
to
medium
antibiotics are used and linked
so that they repress the
expression of the gene of our
interest. As the selection
pressure is removed, plasmids
which have the repressors for
gene of interest are lost, thus
revealing the gene on using the
correct, unique series of
antibiotic washes.
Differential Growth Rates
Fluorescent Imaging
Fig 2.1: Segregational
instability during cell
division
Fig 2.2: Differential growth
rates in presence and absence
of plasmid due to excess
metabolic strain.
Fig 2.3: Directed plasmid loss
based on order of growth
conditions.
Fig 5: Experiment
to compare growth
rates of various
strains in different
antibiotic media.
Differential Growth Rates - Results
Fig 7.1: Experimental procedure for measuring the
rate of plasmid loss by fluorescent imaging.
Circuit
Fig 7.2: Transformed cells with RFP, CFP and RFP–CFP
(co-transformants) seen under DIC, red filter and cyan
filter.
Fluorescent Imaging -Results
P1
P2
Fig 8. Fluorescent imaging
under different conditions
demonstrating plasmid loss
P3
Fig 3.1 General circuit for 3 plasmid system with code length
of 2 units Ab2Ab1. The cells are grown first in Ab2 and then
in Ab1.To maintain the whole system, the cells are grown Ab3.
Fig 3.3: General circuit for 2 plasmid system with code length
of 1 unit – Ab1.The system is maintained by growing in Ab2
Fig 3.2:The 3 plasmid system.
Fig 3.4: The 2 plasmid system.
Fig 6.1: a) DH5a; b) RFP (pSB1C3); c) CFP (pSB1A2); d) RFP (pSB1C3) - CFP
(pSB1A2) grown in LB, LB+Amp, LB+Chl, LB+Amp+Chl.
Proof of concept experiments
Fig 3.5: K272001- RFP constitutive
expression cassette
Fig 3.6: K272002- CFP constitutive
expression cassette
Fig 6.3: All 4 strains in
their corresponding
antibiotic media (OD and
log(OD) respectively versus
time)
Fig 6.2: All 4 strains in media without
antibiotics (OD and log(OD) respectively
versus time)
Key: From top to bottom
1. RFP in Chl
2. RFP in No Ab
3. CFP in Amp
4. CFP in No Ab
5. RFP+CFP in Chl+Amp
6. RFP+CFP in Amp
7. RFP+CFP in Chl
8. RFP+CFP in No Ab
In each individual cumulative
distribution plot:
X axis: Normalized
fluorescence intensity
values
Y axis: Fraction of total
population
time
Caveat: No specific trend is observed in the case of cyan fluorescence, as the expression levels of
CFP are low and there is considerable cellular auto fluorescence which makes signal insignificant
above the background noise.
Expected Behavior
Conclusion
The differential growth rate curves suggest the following:
•In media with no antibiotic(s) (Figs 6.2):
DH5α cells show a higher growth rate in the exponential phase compared to
the cells with either or both plasmids. This indicates that the cells which have either
or both plasmids are under a higher metabolic strain than the ones with no plasmid.
Fig 4.1: :2 plasmid system demonstrating cell fate when
grown in Chl followed by Amp.
Fig 4.2: :2-plasmid system demonstrating cell fate when
grown in Amp followed by Chl.
THE TEAM:
Abdul Majeed
Ajit Kamath
Arun Murali
•In media with corresponding antibiotic(s) (Figs 6.3):
Cells with no plasmid (DH5α) show a higher growth rate than cells with
one plasmid and those with two plasmids. Ultimately, all the cells reach the same
saturation value. It is the time to reach this saturation value which differs in each
case.
INSTRUCTORS:
Bhavya B
Harshvardhan
Pawan Kumar
Ramakrishna
Srivats V
Swathi Ayloo
Dr. Guhan Jayaraman
Dr. Madhulika Dixit
Dr. Mukund Thattai
SPONSORS:
The fluorescent imaging experiments suggest the following:
•As expected, the co-transformed cells show the loss of RFP containing plasmid under
conditions which exert no selective pressure on the cells to retain it.
(Refer to Figure 8: Rows 6 and 8 in comparison to Rows 5 and 7).
•The fraction of cells which show low intensity of fluorescence in presence of
chloramphenicol is considerably lower than those which are grown in no antibiotic.
The concept of plasmid loss works and we have successfully demonstrated by our
proof-of-concept experiment.
REFERENCES:
IIT-Madras iGEM Wiki 2009
< http://2009.igem.org/Team:IIT_Madras >