Part:BBa_K4907123

pHrpL-B0030-gfp-B0015

Biology

pHrpL

pHrpL(BBa_K4907019) 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 hrpL promoter will not be activated by HrpR (BBa_K4907021) or HrpS (BBa_K4907022) 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).

gfp

The green fluorescent protein (GFP) BBa_K4907036 is a protein that exhibits bright green fluorescence. Its excitation wavelength is 488 nm, and its emission wavelength is 533 nm. When gfp is expressed in bacteria, it produces non-exocytosed GFP proteins that cause the bacteria to fluoresce green at an excitation wavelength of 488 nm. It is a commonly used reporter for expressing and tracing.

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 hrpL promoter 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. If we use CspA Cold-responsive elements to express proteins at low temperature, there will be a risk of gene leakage at a higher temperature than we expected because the response temperature of it 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. 1). Under low-temperature conditions, only when both proteins are expressed, then can the expression of downstream genes be induced, reducing the leaky expression. Fig. 2 Gene circuits of hrp system regulated by the CspA CRE

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit of composite part BBa_K4907123, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (1088 bp) can be observed at the position between 1000 and 2000 bp (Fig. 3).

Fig. 3 DNA gel electrophoresis of the colony PCR products of BBa_K4907123_pSB3K3.

Double transformation

We used double 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 </i>E. coli</i> 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 P_hrpL will be activated when HrpR and HrpS are both expressed, but it will not be activated by HrpR (BBa_K4907021) or HrpS (BBa_K4907022) alone.

Fig. 4 The results of immunofluorescence 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
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 881