Part:BBa_K3071023

Co-activator construct for activating synthetic clp-regulated pspA promoter with sequencing part

Description

This composite part contains the two translational units that encode the two proteins that encode the components for activating the pspA promotor include the sigma-54 RNA polymerase cofactor (BBa_K3071021) and Clp-pspF fusion transcriptional activator (BBa_K3071020). The information encoding for the two related protein is transcribed as one mRNA strand in this design. This part is one of the three parts used to construct our detection system in our part collection, the other two parts include (BBa_K30710022) and (BBa_K3071024).

Usage

Figure 1 illustration of our synthetic biological system upon DSF activation

The responsible sensor kinases system detecting DSF in the environment are the RpfC-RpfG. After a series of auto-phosphorylation in RpfC, phosphotransfer from RpfC to RpfG occurs. This leads to the activation of the phosphodiesterase domain in the RpfG, which involves the degradation of the second messenger cyclic-di-GMP. As one of the downstream results, Clp binds to the target CBS I and CBS II. (Figure 1) Without the DSF induction, cyclic di-GMP will not be degraded but binds to the Clp. The binding between Clp and c-di-GMP prevents Clp from binding the CBS.

Using the RpfC-RpfG system as the foundation, we fused the PspF protein with Clp to enhance the binding specificity and prevent expression leakage. The DNA binding domain in the C-terminus of pspF (BBa_K3071006) is replaced by the Clp to construct the fusion transcription activator for our reporter construct (BBa_K3071024). This transcription activator can activate the CBSI & II-regulated pspA promoter (BBa_K3071014) upon the diffusible signal factor (DSF) appears.

pspA promotor is a sigma-54 (σ-54) regulated activator dependent promotor. sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this promotor, which can be activated in E. coli by the bacterial enhancer-binding protein PspF (BBa_K3071006). PspF transcription activation domain (pspF TAD) functions by binding to the upstream activation sequences (UAS) near the promoter and contacting the promotor-bound σ-54 RNAP via DNA looping stabilized by the binding of integration host factor (IHF). Previous research has demonstrated the property of pspF-dependent and enhancer-specific transcription activation of pspA promotor.

Activation of PspA promoter leads to mRNA expression of the reporter eforRed chromoprotein as an output signal after detecting the existence of DSF.

Characterization

Figure 3 Western blot analysis of pspF-Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000)

Western Blot data shows that this construct is appliable to correctly express our candidate proteins under IPTG-induced T7 promotor activity. Clone was induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, pspF TAD-Clp was confirmed with successful expression at all time points. Results of blots probing Clp-pspF showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.

Figure 4 rt-qPCR data Azathioprine treatment on the eforRED mRNA expression level

Azathioprine is an indirect supressor to cyclic-di-GMP, treatment of azathioprine to bacteria culture can reduce the cyclic-di-GMP level and lead to activation of the pspF-Clp and by-pass the RpfC/RpfG two-component system.The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA.

Figure 5 rt-qPCR data DSF treatment on the eforRED mRNA expression level

The result from rt-qPCR data shows that the above sysntehtic biological system is functional with significant up-regulation of reporter mRNA upon DSF activation. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 953
  
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  Illegal XbaI site found at 953
  
• 25
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  Illegal XbaI site found at 953
  Illegal NgoMIV site found at 1031
  Illegal AgeI site found at 136
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• 1000
  COMPATIBLE WITH RFC[1000]