Team:XJTU-China/Engineering
Engineering Success
Toggle Switch
1. General Design
This circuit is an updated version of the traditional toggle switch developed by James J. Collins's group in 2000 (Gardner et al. Nature, 2000), which contains two feedback each controls the other, and can achieve bistability of protein expression in different inducing conditions.
Two promoter-repressor systems (lacUV5 promoter-lacI and lambda pomoter-CI857) with sfGFP and mRFP respectively, was constructed in our updated version, to monitor the two states of the circuit. With induction of IPTG, the downstream genes of lacUV5, that is, CI protein and mRFP will expressed, while those in the downstream of lambda promoter (lacI and sfGFP) will be repressed. Even without IPTG induction after several hours, the lack of lacI expression will result in the stability of red fluorescence. At temperatures above 42 ℃, gene expression will be flipped into another state, the stable expression of lacI and sfGFP, and the state will maintain even without heat. The GFP and RFP can be altered with other functional genes such as tyrptohan synthesitic genes to achieve the bistable expression and synthesis of tyrptohan.
The design of toggle-switch circuit is shown in Fig. 1.1.
Fig. 1.1 Design of Toggle-Switch
Circuit
2. in-silico Engineering
2.1 Design, Build and Test
Before our experiment, modeling was performed for in-silico prediction of behaviours of the toggle switch circuit. We constructed a equation set based on transcription and expression to describe the changes in the relative expression levels of GFP and RFP under different inducing conditions (IPTG induction or 42℃ heat). Equations used are listed in Fig. 2.1
The in silico result of the expression level of sfGFP and mRFP under different leaky expression factor α0 as shown in Fig2.2， which indicates that the behavior of toggle switch is largely affected by the leaky expression of two promoters. A bistability can be achieved under low α0, and toggle switch will fail in case of high α0.
Fig. 2.2 Toggle Switch Modelling
Result.
IPTG is
introduced at 1000 min
and the induction condition is switched to 42℃ at 2000 min.
Fig. 2.1 Toggle Switch Modelling
Equations.
For detail, see also our Modelling
2.2 Learn
With the result of our in-silico prediction, we concluded that reduced leaky expression of promoters is the key to construct the toggle-switch circuit. This would provide guidance for our following experimental design, including selection of the RBSs with appropriate strength to weaken the possible leaky expressions.
3. Experimental Engineering
3.1 Design
Generally, the toggle-switch circuit is constructed utilizing two sets of promoter-repressor system, namely, lacUV5 promoter-LacI and lambda promoter-CI857, in addition to sfGFP and mRFP as the reporter genes (see the Fig. 1.1). Promoter lacUV5 initiates the transcription of the downstream genes cI857 and mRFP, leading to a combined result of red fluorescence and pλ inhibition. While the pλ, just the contrary, starts the expression of GFP and lacUV5 repressor LacI. Thus, if inducing the cells with IPTG, the inhibition on lacUV5 by LacI will be released, triggering the generation of red signal. And the treatment of higher incubation temperature, like 42℃, promotes the degradation of cI protein, inducing the biological function of pλ then causing a green signal.
3.2 Build
In our project, Golden Gate Assembly (GG) is applied to ligate all the genes and their backbones pET28a+. Thus, a series of DNA fragments with flanking sequences which are consistent type IIS restriction enzyme recognition sites and complementary restriction sites are generated using PCR amplification. Additionally, the 5' overhang of primers may also extend the amplicons with some short but fundamental parts like Shine-Dalgarno sequences, promoters and terminators.
The figure of agarose gel electrophoresis of DNA fragment that build the toggle-switch plasmid is shown in Fig. 3.1(a).
Fig. 3.1 Agarose gel electrophoresis of
toggle-switch circuit. (a) The circuit of toggle switch (3735bp) is
constructed and the electrophoresis result shows the expected band (right lane).
(b) The toggle-switch plasmid has the length of 5920bp. Then the plasmid is
subjected to double
digestion with XbaI and SapI to yield fragments in 3825bp and 2095bp
These two fragments are consequently used for GG to construct the plasmid containing our toggle-switch circuit. After transformation to E.coli DH5alpha, plasmid extraction and a set of screening including colony PCR and double digestion, strains with expected plasmid are selected, indicating the toggle-switch circuit is built. And the sequencing result confirms no mutation in the circuit.
3.3 Test
3.3.1 RT-qPCR quantifications of Toggle-Switch
We first used RT-qPCR to detect the transcription of each gene under different induction conditions, so as to preliminarily judge the effect of promoter bistable expression.
Method:
Cultivation: Using LB liquid medium, 37℃, 200rpm. After
cultivating for 3h,
cells
are
cultivated under inducing conditions (1mM IPTG / 42℃) for 8h
respectively.
Total RNA extraction: Using RNAsimple Total RNA Kit,DP419 (TIANGEN
BIOTECH (BEIJING)
CO.,LTD.)
cDNA preparation: Using Evo M-MLV RT Mix (Vazyme Biotech Co.,Ltd);
template
concentration: 50ng RNA/ul; reaction condition: 37℃ 15min,
85℃
15sec.
qPCR: Using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd).
Relative Normalized Expression data is calculated by using the equation
below,
Relative Expression = 2-[ΔCt(T)-ΔCt(C)]
where ΔCt(T) represents the difference between
Ct value of target gene and internal standard
gene in
treatment
group; ΔCt(C) represents the difference between
Ct value of target gene and internal standard
gene in negative
control group.
Results and Discussion: Cells were induced by two inducers IPTG and 42℃ separately, to test the two state of toggle switch under different conditions. As shown in Fig. 3.3, with IPTG induction, the relative transcriptional level of mRFP was significantly increased about 5 folds in the steady state (a), and cI857, that is under the same control of lacUV5 promoter with mRFP, also showed the improved transcriptional level (b), indicating one state promoted by lacUV5 promoter. In contrast, when cells were incubated in 42℃, the mRNA level of sfGFP was remarkably enhanced from 1.5x105 to 7.9x106, indicating another steady state controlled by lamda promoter. The transcription of downstream LacI was just slightly increased as shown in Fig. 3.3(b), which may attribute to the copy of lacI gene in the genome of E.coli. In all, induction by 1mM IPTG resulted in obvious up-regulation of transcription of mRFP and cI857, while inhibition in sfGFP and LacI. And under 42℃ induction, the relative expression of genes has been reversed. The results indicate the two steady states can be achieved under two inducers respectively.
Fig. 3.3 Relative normalized expression
of
(a): mRFP and sfGFP (b): cI857 and lacI in toggle-switch circuit measured by
RT-qPCR
3.3.2 Characterization of Bistability of Toogle-Switch
Method:
Due to the expression of
reporter genes in toggle
switch, GFP
and
RFP
signals are detected by a microplate reader (SpectraMax i3). After
normalization,
that is, dividing the fluorescent signals by corresponding OD600, the intensity of
fluor-signal per cell is gained. And upon inducing with either IPTG or
42℃,
temporal
determination of all the OD600, GFP &RFP
fluorescent
signals provides us a set of
data that could describe the bistable-switch function of our
toggle-switch
circuit.
We used full spectrum scanning to determine the most appropriate
excitation and
emission wavelengths for the two fluorescent proteins. Wavelength used
are as
below:
sfGFP: Excitation 485nm; Emission 535nm
mRFP: Excitation 587nm; Emission 627nm
Cultivation: Using LB liquid medium, 37℃, 200rpm.
Induction: 1mM IPTG was used in 0-719 min culturing. Then the cells were
collected
by centrifugation and replaced with fresh medium without IPTG, and
cultured at
42℃
The obtained fluorescence intensity data was normalized by subtracting the intensity of the negative control (DH5alpha strain containing empty pET8a+ vector) and divided by the 600nm absorbance (representing the thallus concentration).
Result:
The bistability of toggle switch was tested by switch of two inducers. As shown in
Fig. 3.4, cells were first induced by IPTG, and mRFP was expressed showing stably
increased red fluorescence. Because of the cI857 was highly expressed in the same
time, lamda promoter was inhibited and little expression of sfGFP was observed. At
around 720 min, cells were washed, resuspended in fresh LB medium, and incubated in
42℃. The change of induction condition led to the shifted expression of sfGFP
and
LacI, and the expression of mRFP was dramatically decreased due to the inhibitory
effect of Lac I on lacUV5 promoter. Fig. 3.4 clearly showed the fast switch of two states
controlled by two inducers, and the bistability can be aslo achieved under each
conditions.
Fig. 3.4 Relative fluorescence
intensity
of sfGFP and mRFP in toggle-switch circuit under different inducing
conditions
3.4 Learn
Conclusion: We confirmed the function of toggle-switch circuit in RT-qPCR and fluorescence measurement results. We successfully achieved the bistable expression of sfGFP and mRFP under different induction conditions. The alternation of the two fluorescence intensities was observed after the change of conditions, reporting the intensities of respective promoters.
According to the above results,the feasibility has been proved that we can use this circuit to control the expression of aroG, trpBA and pykA genes, so as to control the cell into different states of “proliferation” and “production”.
Read more results of our new parts at Proof of Concept or improvement.
For protocols we apply in our labwork, see also: Protocols