Part:BBa_K1315007

The BCAM0228 Response Regulator from Burkholderia

This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia spp. It is suggested that it activates the cblD promoter(BBa_K1315008).

Usage and Biology

The Burkholderia diffusible signal factor (BDSF/cis-2-dodecenoic acid) is a signalling molecule that regulates virulence in Burkholderia cenocepacia. BDSF belongs to the DSF family which was first described in Xanthomonas campestris. BCAM0227 has been identified as a sensor kinase of the BCAM0227-BCAM0228 system that picks up BDSF.

However, the confirmed BDSF signalling pathway only goes as far as BCAM0227. It has been proposed that the next steps involve the BCAM0228 regulator protein. The BCAM0228 regulator though is more similar to the OmpR two-component response regulator rather the RpfG(BBa_K1315003) one. Thus, BCAM0228 is the hypothetical regulator of the BCAM0227-BCAM0228 system.

http://www.ncbi.nlm.nih.gov/pubmed/21227698 http://www.ncbi.nlm.nih.gov/pubmed/?term=A+sensor+kinase+recognizing+the+cell-cell+signal+BDSF+(cis-2-dodecenoic+acid)+regulates+virulence+in+Burkholderia+cenocepacia.

B

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 18
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 226
  Illegal NgoMIV site found at 326
  Illegal NgoMIV site found at 370
  Illegal NgoMIV site found at 598
  Illegal NgoMIV site found at 728
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

direction	Forward
function	Two-component Regulator

BCAM0228 is designed to be used with BBa_K1315008, which is the cblD promoter region, and the sensor kinase BCAM2207 (made by iGEM Dundee as a synthetic gene that could not be BioBricked at this time). BCAM0228 was epitop etagged with HA and expressed in an E. coli chassis (Dundee Figure 1). However, the response regulator was found to activate BBa_K1315008 transcription without inducer and without sensor kinase - see the experience pages of BBa_K1315008 . It is possible an endogenous kinase or chemical is phosphorlylating BCAM0228 in E. coli. Troubleshooting is required.

Dundee 2014 Figure 1 Western blot of production of HA-tagged BCAM0228 (and BCAM0227)