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Latest revision as of 20:00, 28 October 2013

Hybrid Promoter for Band-pass Filter

hybrid promoter for the band-pass filter

Construction of our hybrid promoter

We modified a bacteriophage ϕR73’s P₂ promoter into a hybrid promoter that can be activated by the ϕR73δ activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We constructed the hybrid promoter by replacing the sequence between position -1 and -25 of P₂ promoter with the cI binding site OR1 from Phage λ P_R promoter. When ϕR73δ activator binds to its target sequence upstream of -35 element of the hybrid promoter, the transcription will start. The binding of cI dimers downstream of -35 element will block the binding of σ⁷⁰ factors and thus repress the transcription even when ϕR73δ is bound. (Fig. 1).

Figure.1 Construction of Our Hybrid Promoter.
Sequence information of phage φR73 P₂ promoter (a), phage λ P_R promoter (b) and our hybrid promoter (c) are shown. a, In the P₂ promoter, φR73δ binds to a region between position -42 and -71 and activates transcription. b, In P_R promoter, cI dimer binds to OR1 site (marked as blue) between position -9 and -25, blocking binding of σ⁷⁰ factors and inhibiting transcription. cI binding region indicates the sequence we used to replace the corresponding region in P₂ promoter.c, The hybrid promoter is constructed by replacing sequence between position -1 and -25 of φR73 P₂ promoter with sequence at the same position in phage λ P_R promoter that contains an OR1 site. The hybrid promoter is co-regulated by φR73δ and cI, with φR73δ activating and cI repressing. The repression of cI dominates over the activation of φR73δ, since the steric hindrance created by cI dimer prevents formation of transcription initiation complex even when RNA polymerases are recruited through the help of φR73δ.

Sequence and Features

Characterization

To comprehensively characterize the dynamic performance of the hybrid promoter, we put two regulators of the hybrid promoter, ϕR73δ and cI, under the control of two different inducible promoters, P_sal promoter and P_tac promoter. (Fig.2). This enables us to manipulate separately the expression levels of two regulatory proteins through tuning P_sal and P_tac promoter by adding different concentration combinations of inducers (salicylic acid for P_sal promoter and IPTG for P_tac promoter).

Figure.2 Testing construct for hybrid promoter.
φR73δ was put under the regulation of P_sal promoter and cI was put under the control of P_tac promoter. Salicylic acid (SaA) will induce φR73δ expression and activate the hybrid promoter. Isopropyl β-D-1-thiogalactopyranoside (IPTG) will induce cI expression and repress the hybrid promoter. Expression level of the two regulatory proteins can be manipulated separately by adding different concentration combinations of SaA and IPTG.

To comprehensively characterized the hybrid promoter's transcription activity, we exposed the characterization circuit (Fig.2) to a 8x8 two-dimensional induction assay established by combining 8 different concentrations of salicylic acid and 8 different concentrations of IPTG and measured the fluorescence intensity of sfGFP reporter using Flow Cytometry. (Fig.3)

Figure.3 Characterization of hybrid promoter's dynamic performance.
A two-dimensional inducer concentration assay was established by combining 8 different SaA concentrations (0, 0.1, 0.5, 1, 5, 10, 50 and 100µM) with 8 different IPTG concentrations (0, 1, 10, 50, 100, 150, 200 and 300µM). Bacteria cells expressing the testing construct were exposed to the assay and sfGFP fluorescence intensity was measured using Flow Cytometry. For a fixed IPTG concentration, fluorescence intensity gradually increased as SaA concentration increased. For a fixed SaA concentration , fluorescence intensity gradually decreased as IPTG concentration increased. These features indicated that the promoter functioned as expected.

The hybrid promoter worked as expected. For a fixed IPTG concentration, the sfGFP fluorescence gradually increased as the salicylic acid concentration increased, exhibiting a Hill-function type dose-response curve. For a fixed high salicylic acid concentration under which sfGFP expression is visibly induced, the fluorescence gradually decreased as the IPTG concentration increased, also exhibiting a Hill-function type dose-response curve. These data prove that the hybrid promoter can indeed be activated by ϕR73δ and repressed by cI, and the repressing effect of cI protein dominates over the activating effect of ϕR73δ protein, because transcription of the hybrid promoter can still be repressed to a very low level by cI even when ϕR73δ is expressed at a very high level.

We hypothesized that the negative feed-forward loop in the testing construct may actually represent a transformed version of the negative loop in the original Band-pass Filter construct. So we fit our model to the data from the testing construct in order to get real parameters for the Band-pass Filter circuit. (Fig.4)

Figure.4 Model based data fitting for φR73δ activator and cI repressor.
a, Experimental points are sfGFP fluorescence intensities under different SaA concentrations without IPTG. The model based fitting curve provided parameter values for n_A' and K_AG. b, Experimental points are sfGFP fluorescence intensities under different IPTG concentrations along with 100µM SaA. Model based data fitting curve provided parameter values for n_B', K_BG and k_AG•k_BG. Fitting results: n_A'=0.72705; K_AG=62.99928; n_B'=1.15498; K_BG=11.53988; k_AG•k_BG=24671.78415. Definitions for the parameters can be viewed in equations written in Model page.

To test the activation effect of φR73δ, we constructed an activation circuit adopting Psal/NahR biosensor to regulate the expression of φR73δ. When exposed to inducers of NahR, Psal is initiated thus φR73δ is expressed. If φR73δ indeed activates the hybrid promoter, it is expected that the fluorescence intensity changes along with the change of inducers' concentration.

Figure.5 Construction of activation circuit and corresponding data.
Left: Construction of activation circuit of Band-pass Filter. φR73δ is located on low-copy backbone pSB4K5 to avoid toxicity accumulation.
Right: Effect of activation circuit of Band-pass Filter. Horizontal axis stands for different concentrations of NahR inducer 4-MeSaA. Vertical axis represents induction ratio. It is obvious that φR74δ indeed activates the hybrid promoter, thus initiates the expression of sfGFP.

After confirming the activation effect of φR73δ, we then tested the inhibition effect of CⅠ protein to the hybrid promoter. A high basal level of fluorescence intensity was constructed via constitutive expression of φR73δ. Expression of CⅠ protein is regulated by inducer of NahR under Psal promoter. When exposed to inducers, the expression of CⅠ is supposed to inhibit the expression of reporter sfGFP if the hybrid promoter is indeed negatively regulated by CⅠ.

Figure.6 Construction of inhibition circuit and corresponding data.
Left: Construction of inhibition circuit of Band-pass Filter, consisting of constitutive φR73δ, inducible CⅠ protein and reporter gene sfGFP under the control of hybrid promoter.
Right: Effect of inhibition circuit of Band-pass Filter. Horizontal axis stands for different concentrations of NahR inducer 4-MeSaA. Vertical axis represents induction ratio. It is obvious that CⅠ indeed inhibits the hybrid promoter, thus reduces the expression of sfGFP.