Part:BBa_K3740062

J23100-B0034-fcsR-rrnB T1

Description

This is a generator of fcsR, that is used to regulate the level of c-di-GMP.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 766
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 124
  Illegal NgoMIV site found at 869
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 763
  Illegal BsaI.rc site found at 511
  Illegal SapI.rc site found at 876
  

2021 SZPT-China

Biology

This is a generator of fcsR (BBa_K3740022), that is used to regulate the level of c-di-GMP.

Usage

The coding sequences for FcsR were inserted into the expression vector with BBa_K880005 (BBa_J23100 & BBa_B0034) to obtain J23100-B0034-fcsR-rrnB T1 (BBa_K3740062). 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 J23100-B0034-fcsR-rrnB T1.

Characterization

1. Identification

As shown in Figure 2, the generator was identified successfully by PCR amplification.

Figure 2. Agarose gel electrophoresis image of J23100-B0034-fcsR-rrnB T1. BBa_K3740062, 975bp

2. Characterization

As shown in Figure 3, BC production in J23100-B0034-fcsR-rrnB T1-pSEVA331-G. hansenii ATCC 53582 was lower than the control group pSEVA331-G. hansenii ATCC 53582, indicating that FcsR was capable of hydrolyzing c-di-GMP in G. hansenii.

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