Engineering Success
Toggle Switch

2. in-silico Engineering

2.1 Design, Build and Test

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).

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.

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.

3.3.2 Characterization of Bistability of Toogle-Switch

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.

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”.

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