Team:SCU China/Repressilator/Repressilator
Repressilator is the
necessary
and basic part of our project, which ‘grant’ the four melatonin-synthetic enzymes
(actually COMT) periodically expression pattern.
Repressilator was first construct by Michael B.
Elowitz & Stanislas Leibler in 2000. As
a well-characterized synthetic circuit, it is designed to help understand naturally
occurring oscillating networks and remarkably precise and robust dynamics shown in
many
biological systems, for example, the circadian clock.
Just like the name ‘repressilator’ indicate, the circuit actually is built from the
combination of (three) transcriptional repressor systems that are not part of any
natural biological clock, they inhibit mutually to achieve an oscillating pattern,
(A B
C A); in other words, each of the downstream gene is controlled by the previous
repressors. The protein half life of the repressors and the induction threshold of
the
promoter mainly contribute to the oscillating pattern and period.
In the original ones the reporter is built in
another plasmid and co-transform into the
E.coli. pSC101 is choosed for the backbone because of its low replicating number. In
2016 researchers do some modification work for the both plasmid and E.coli starin.
Reporters are inserted into the repressilator plasmid and fuction simutanously as
trition sponge because triple reporters have exactly the same promoters of
repressors
elongating the period of reaching induction threshold for the repressors and
diluting
repressor effective concentration referring to the number of proteins binding with
the
operator sequence in the promoter.(Fig1)
To some extent, original repressilator can exhibit a wide range of dynamical features but often with lower accuracy, especially obvious unsynchrony and phase drift. In 2016, researchers modified the original one and rebuilt a triple reporter repressilator without repressor degradation (E.coli proteaseΔclpXP strains) and with titration sponge. It was reported that cells could display macroscopic, population-scale oscillations and shew a typical 14 generation cyclic period without cell–cell communication. In other words they can synchronize automatically in a liquid culture and maintain it after proper (1mM) iPTG induction, which provides a theoretically feasible design to combine the oscillating network with melatonin synthesis via TetR controlled promoter of key enzyme COMT. Thus, in this part, we design a lot of experiments to verify the repressilator basic properties in different conditions and try to acquire a more precise and regular oscillating pattern as the origin of the periodic signal.
We bought well-construct
commercialized plasmid pLPT107 (as reported in the paper) (Fig1)and transformed it
into
E.coli MC4100 and DHL708ΔclpXP strain (received from team “Peking”, more details
about
knock-out are listed at the end) (both are LacI knocked out) respectively to test
the
function of protease for oscillating period and phase drift, because protease
knocking-out may have some potential influence on the enzyme activity and kinetics
of
whole four-enzyme system due to the protein half-life changes and it is better not
to
use this strain in enzyme expression if not necessary. The real-time fluorescence
intensity of all three reporters CFP (400/476nm) YFP(480/530nm) RFP(590/630nm) is
measured by 37℃ culturing the strains in 96-well plate in plate reader. And every
90mins
bacteria is diluted to OD0.1-0.2 to keep in early exponential phase. We calculate
the
fluorescence intensity/OD600 in the figure to represent the fluorescence protein
concentration in a single unit such as one E.coli cell. And our results shows that
strains obey a 120min long oscillating period, whileΔclpXP strain exhibits a more
regular oscillating pattern than its counterpart, whereas it is quite confused that
the
period is such short and DHL708ΔclpXP only differ with MC4100 in amplitude not in
the
period length which is inconsistent with the paper (Fig 2). However DHL708 exhibits
colony ring-structure comparing to MC4100 (Fig3), and all indicts that protease
knocked-out strain is necessary for following experiments to achieve a much more
regular
cyclic pattern.
Figure 2. Fluorescence intensity changes in the whole measuring period. (a) CFP and (b)YFP fluorescence change in a oscillating pattern for both MC4100 and DHL708ΔclpXP strains in a period of approximately 120mins. The major difference between protease clpXP knout-out and normal MC4100 is MC4100 shows a higher and more irregular oscillating amplitude especially at the begin(0-200min) and at the end (1500-1675min). (c), and for RFP both strains oscillates in a similar low level 100-300 A.U./OD 1300mins before, but after 1300min the fluorescence intensity of MC4100 increase obviously and even reaches 5000 A.U./OD in the following 300min which cannot be illustrated reasonably.
Figure 3 colony ring structure.a-c, A 3-mm diameter colony of DHL708ΔclpXP cells with the triple reporter repressilator (pLPT107) reveals tree-like ring patterns in fluorescent protein levels. e-f, However colony of MC4100 cells with the triple reporter repressilator (pLPT107) do not show ring-pattern. The images are acquired using Leica microscopy and solid culture medium are conditioned on the slides for bacterial colony incubation and image acquisition.
To optimize the oscillating period, we try to add additive iPTG in the measuring process to urge the cell synchronize under the outer ‘pressure’. And different concentration of iPTG is added into the dilution M9 medium to achieve ultimate 100/250/500/1000μM. We find that additive iPTG could help to synchronize in a more robust way by amplify the oscillating amplitude, in other word the fluorescence intensity changes more rapidly.Another finding is that with the iPTG concentration increase, the induction effect increase firstly and then decrease and 500uM iPTG is the best. (Fig 4)
Generally, the periods of synthetic circuits depend on so many different parameters that change with conditions, as conditions change, the plasmid copy numbers, RNA degradation, gene expression, and cell volume change in non-trivial ways, making it robust relative to internal physiological time scales such as the generation times. So we utilized different culture conditions (measured by additive LB ratio-5% 10% 20% 40%, higher ratio means richer nutrition) to test whether cyclic period will change with the increase/decrease of the nutrition and on the other hand try to get a longer oscillating period. Because we do hope this period could be adjusted closer to human circadian clock (12 hours) so we can express melatonin in this pattern to imitate the natural process. (Fig 5) MC4100 and DHL708ΔclpXP continuously incubate in 37 ℃ plate reader in different M9 culture medium and RFP fluorescence is measured in these conditions. Oscillating pattern do not exhibit significant difference among various M9 medium for DHL708ΔclpXP, which indicts that culture conditions, especially nutrition, have no obvious influence on oscillation. And it is possible for us to rhythmic express our melatonin synthase in the E.coli and keep a regular period.
Next, in order to investigate whether the strains or bacteria have possibility to colonize into the gut environment and change their oscillating period, artificial intestinal fluid (formula in method) was applied to substitute the normal culture medium and different ratio of LB was added to mimic the various nutrition state for a person in a day (Empty to Full).As Fig 6a indicts both MC4100 and DHL708 are able to colonize stably in the artificial intestinal fluid and Fig6b suggests that both strains can maintain their oscillating period in artificial intestinal fluid while DHL708ΔclpXP could keep a much better regularity in various nutritional intestinal fluid simultaneously.
The OD of all experimental groups increase exclusively relative to different culture conditions, as indicts, absolute LB and intestinal fluid represent the fastest and slowest growth rate respectively and DHL708ΔclpXP cells generally possess a slower growth rate referring to its counterparts during the whole measurement. b, both strains can maintain their oscillating period in artificial intestinal fluid while DHL708ΔclpXP could keep a much better regularity in various nutritional intestinal fluid simultaneously. On the other perspective, the nutrition of the fluid nearly exhibit no obvious impact on oscillation, no matter period and pattern.
ClpXP protein is a protease which can recognize
ssrA
tag, a degradation tag, and target the attached protein for destruction, belonging to the heat shock protein Hsp100
family.
As the first repressilator reported in 2000,
ssrA
tag is added to repressors in
repressilator in order to bring the effective repressor protein lifetimes to that of
mRNA.
However, the research in 2016 showed that the
ssrA
tag induced ‘retroactivity’ effects
on oscillations owing to competition for shared proteases, and removing this
interference created very regular oscillations, with longer periods.
Hence we decide to knock out the clpXP to
create
a
more regular oscillation with
longer periods.
Because the pKD3 plasmid we have was wrong, we
use
one part of Red recombination system.
The biggest difference between our method and the traditional method is that our PCR
production doesn’t have FRT site so we don’t need to transform the pCP20 plasmid
into
the bacterial to remove the reporter gene. Our RED recombination system utilize the
pKD46 plasmid to express three proteins: Exo can produce 3’-overhang, Bet can induce
two
single strand DNA molecule becoming double strand DNA and Gam can inhibit the
function
of RecBCD protein. The pKD46 plasmid is then transformed into E.coli, with these
three
proteins expressing under the induction of L-Arabinose. Then the PCR production of
reporter gene, Kanamycin resistance gene, which is flanked by two 57bp homologous
arm of
clpXP, is transformed into the bacteria expressiong Exo, Bet and Gam by
electroporation,
leading to the substitution of Kanamycin resistance gene for clpXP. Finally, the
pKD46
plasmid will be lost by high temperature culture, and the ΔclpXP E.coli is screened
by
Kanamycin.(Fig 7)
Because our reporter gene is Kanamycin resistance gene, the bacteria is plated on the solid medium contained Kanamycin. The number of colonies between experimental group is much larger than that of the control group, which is E.coli with pKD46 without the insert bringing in the homology arms. The experimental group is cultured in liquid media and then used as template of PCR to confirm the recombination colonies. And then colony PCR is used to confirm the result of recombination. The PCR products from recombination colonies are below 2kb, while the products from wild type is about 2kb. Then we optimized the PCR reaction and sequenced the product from recombination colonies, whose result indicts that we have successfully knocked out the clpXP gene. (Fig.8)
a,The electrophoresis result of PCR production of adding homologous arm to K resistance gene. The result of PCR was accordance with the anticipated objective strap size (1157bp).
b, The result of single bacteria colony PCR after optimization. It’s clear that the length of PCR production is less than 2kb.
c, The result of single bacteria colony PCR after optimization. It’s clear that the length of PCR production is less than 2kb.
d, The result of single bacteria colony PCR. The 12 lane is negative control which undergoes the PCR without template. The 13 lane is positive control using the wild type bacteria as template.
e, The recombination colony is obviously lower than 2000bp and has non-specific band at 1000bp. The wild type’s specific band is at 2000bp and the non-specific band is at 700bp.
1. Elowitz, M. B. & Leibler, S. A synthetic oscillatory network
of transcriptional regulators. Nature 403, 335–338 (2000).
2. Potvin-Trottier, L., Lord, N. D., Vinnicombe, G. & Paulsson, J. Synchronous long-term
oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016).
3. Gottesman, S., Roche, E., Zhou, Y. N. & Sauer, R. T. The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338–1347 (1998).
4. Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).
5. Potvin-Trottier, L., Lord, N. D., Vinnicombe, G. & Paulsson, J. Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016).
