Part:BBa_K3740021

rocR, PA3947, cyclic di-GMP phosphodiesterase

Description

Degradation of c-di-GMP in Gluconacetobacter hansenii ATCC 53582.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 258
  Illegal BamHI site found at 1171
  Illegal XhoI site found at 94
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 895
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 802
  

2021 SZPT-China

Biology

The gene rocR is originated from the genome of Pseudomonas aeruginosa (PAO1). And RocR expressed by rocR is the phosphodiesterase (PDE) of c-di-GMP.

Usage

The encoding sequences for RocR were inserted into the expression vector with BBa_K880005 (BBa_J23100 & BBa_B0034) to obtain J23100-B0034-rocR-rrnB T1 (BBa_K3740065). We introduced the constructed plasmid into E. coli DH5α to verify its successful construction, and then transferred it into G. hansenii ATCC 53582 to verify its function.

Figure 1. Gene circuit of rocR

Characterization

1. Identification

As shown in Figure 2, c-di-GMP phosphodiesterase-encoded genes BBa_K3740065 was identified successfully by PCR amplification.

Figure 2. Agarose gel electrophoresis image of J23100-B0034-rocR-rrnB T1 (BBa_K3740065). BBa_K3740065, 1294bp

2. Characterization

As shown in Figure 3, BC production in J23100-B0034-rocR-rrnB T1-pSEVA331- G. hansenii ATCC 53582 and the control group pSEVA331-G. hansenii ATCC 53582 was not significantly different, indicating that RocR was not capable of hydrolyzing c-di-GMP in G. hansenii ATCC 53582.

Figure 3. BC yield by J23100-B0034-rocR-rrnB T1-pSEVA331-G. hansenii ATCC 53582 and the vehicle control pSEVA331-G. hansenii ATCC 53582.