Composite

Part:BBa_K4907126

Designed by: Linhao Zhang   Group: iGEM23_XMU-China   (2023-09-16)


I0500-B0034-hrpR-B0015-araBAD promoter-B0034-hrpS-B0015


Biology

I0500

BBa_I0500 is an Inducible pBad/araC promoter. pBad is an E. coli promoter that is tightly controlled by:
inducer: L-arabinose.
repressor: AraC acts as the repressor

hrpR

This part(BBa_K4907021) codes for HrpR protein. HrpR protein binds to HrpS protein coded by hrpS (BBa_K4907022) forming a complex and then triggering the transcription of pHrpL(BBa_K4907019). pHrpL is the promoter of HrpL, which is an extracytoplasmic sigma factor regulating the expression of type III secretion system (T3SS) from Pseudomonas syringae pv. tomato DC3000, a plant pathogenic gram-negative bacterium. Specifically, it employs the T3SS to cause disease in tomato and Arabidopsis and to induce the hypersensitive response in nonhost plants. Expression of HrpL is controlled by transcriptional activators HrpR and HrpS (1). The pHrpL will not be activated by HrpR or HrpS alone. But when both HrpR and HrpS exist, they can form a complex that activates the pHrpL and induces the expression of downstream genes. This functions like an AND logic gate (2).

hrpS

This part(BBa_K4907022) codes for HrpS protein. HrpS protein binds to HrpR protein coded by hrpR (BBa_K4907021) forming a complex and then triggering the transcription of pHrpL(BBa_K4907019).

Usage and design

To prove the hrp AND gate can work. We used BBa_I0500 to construct the regulation system and obtained the composite part BBa_K4907124 and BBa_K4907125, which were assembled on the expression vector pSB1C3. And we used the pHrpL and GFP(BBa_K4907036) to construct the reporting system and obtained the composite part BBa_K4907123. Those three composite parts together form the verification system of hrp AND gate (Fig. 1) and corresponding gene circuits were transformed into E. coli DH10β for characterization.

Fig. 1 Gene circuits of verification system for hrp AND gate.


We constructed this composite part to combine hrpR(BBa_K4907021) and hrpS(BBa_K4907022) together(Fig. 2).

Fig. 2 Gene circuits of composite part BBa_K4907126.


If we use CspA Cold-responsive elements to express proteins at low temperatures, there will be a risk of gene leakage at a higher temperature than we expected because the response temperature has a broad range. To solve this problem, we plan to combine a logic AND gate with the CspA CRE. Based on it, we designed an AND gate to respond to low temperature, namely, hrp AND gate. In this system, the hrpR and hrpS genes are regulated by the CspA CRE (Fig. 3). Under low-temperature conditions, only when both proteins are expressed, can the expression of downstream genes be induced, reducing the leaky expression.

Fig. 3 Gene circuits of hrp system regulated by the CspA CRE.

Characterization

Dual-plasmid system transformation

We used dual-plasmid system transformation to prove the hrp AND gate. One control and three experimental groups were set up. For R+S group, Plasmid BBa_K4907123_pSB3K3 and plasmid BBa_K4907126_pSB1C3 were transformed into E. coli DH10β which can express HrpR and HrpS. For the remaining two experimental groups, each can only express one of HrpR (BBa_K4907021) and HrpS (BBa_K4907022). As for the control, Plasmid BBa_BBa_K4907123_pSB3K3 and plasmid BBa_BBa_I0500_pSB1C3 were transformed into E. coli DH10β. The positive transformants were selected by kanamycin and chloramphenicol.

Fluorescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of GFP is observed by measuring the GFP Fluorescence/OD600 using microplate reader (Fig. 4). The results of fluorescence showed that the pHrpL will be activated when HrpR and HrpS are both expressed, but it will not be activated by HrpR or HrpS (BBa_K4907022) alone.

Fig. 4 The results of GFP Fluorescence/OD600 to verify that hrp AND Gate can work.

Reference

1. M. Jovanovic, E. Lawton, J. Schumacher, M. Buck, Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system. Fems Microbiology Letters 356, 201-211 (2014).
2. B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 2626
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3559
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 2565
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2400
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2769
    Illegal SapI site found at 961
    Illegal SapI site found at 2382
    Illegal SapI.rc site found at 3402


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