Difference between revisions of "Part:BBa K3832007"

Latest revision as of 19:04, 21 October 2021

Toggle-switch circuit with GFP and RFP

This part 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 promoter-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.

Sequence and Features

1. Useage & Biology

We have constructed a toggle-switch circuit, which can achieve the bistable expression of two transcription units under different inducing condition. We use two groups of promoter-repressor (lacUV5 promoter-lacI and lambda promoter-CI857) to realize the bistable expression. Coding sequences of GFP and RFP are designed downstream the two inducible promoter respectively, which can report the effect of bistable regulation under different induction. The design of toggle-switch circuit is shown in Fig. 1.1.

Fig. 1.1 Design of toggle-switch circuit

2. Modeling

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.

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.

Fig.2.1 Results of modeling on toggle-switch circuit.During 0-1000 min this circuit is not induced;1000-2000 min is induced by IPTG;2000-3000 min is induced by 42℃ heat
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

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. (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.

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: 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 1.5x10^5 to 7.9x10^6, 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): lacI and cI857 in toggle-switch circuit measured by RT-qPCR

3.3.2 Characterization

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).  

Results: 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