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 feedbacks each controls the other, and can achieve bistability of protein expression in different inducing conditions.
Two promoter-repressor systems (lacUV5 promoter-LacI and lambda-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 degrees Celsius, 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, we construct a mathematics model to perform the in-silico test. 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. Equations used are as below.
需要方程图片
Through literature review, we identified partial relevant parameters. We obtained a bistable transformation model under different inducing conditions, and find the leakage expression intensity of two repressors is the decisive factor to determine whether bistable can be achieved, that is, when the leakage expression is not low enough, bistable will fail.
2.2 Learn
By analyzing the result of our in-silico test, we concluded that avoiding leakage expression of repressors is the key to successfully construct the toggle-switch circuit. This would provide guidance for our following experiment design, including selecting the appropriate RBSs to build our circuit.
3. Experiment 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 fragments that build the toggle-switch circuit is shown below.
Fig. 3.1 Agarose gel electrophoresis of
toggle-switch circuit
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.
Fig. 3.2 Agarose gel electrophoresis of
cloned plasmid inserted with toggle-switch circuit
3.3 Test
3.3.1 RT-qPCR
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: The group induced by 1mM IPTG represents obliviously up-regulation of transcription of mRFP and cI857 while inhibited in sfGFP and lacI; under 42℃ relative expression of this genes has been reversed comparing induced by IPTG (Fig.3.3).
Fig. 3.3 Relative normalized expression of
(a): mRFP and sfGFP (b): cI857 and lacI in toggle-switch circuit measured by
RT-qPCR
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:
As in Fig.3.4, the relative fluorescence intensity of sfGFP and mRFP reversed after
changing the induction 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”.