Part:BBa_K1649003:Design

YF1 blue light sensor with GFP reporter

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 605
  Illegal NgoMIV site found at 677
  Illegal NgoMIV site found at 767
  Illegal NgoMIV site found at 785
  Illegal NgoMIV site found at 1297
  Illegal NgoMIV site found at 1590
  Illegal NgoMIV site found at 1684
  Illegal AgeI site found at 319
  Illegal AgeI site found at 1465
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1354
  Illegal BsaI.rc site found at 218
  Illegal BsaI.rc site found at 2904
  

Design Notes

To test the funnction blue light sensor GFP is used to report the function of blue light sensor. The LOV photo-switch YF1 was created by fusing the light perception domain of YtvA to the FixL kinase domain. YtvA acts as a blue-light photoreceptor and carries a blue-light-sensitive flavin-binding LOV domain. The response regulator FixJ forms a two-component system together with FixL.The sensor is inactive under blue light and active in dark.

Measurement

In order to test whether this part works,we cloned this part into pET28a using the NotI and XbaI sites. This plasmid contains a Kanamycin resistance gene and a pBR322 origin. BL21 strains are used to harbor blue light sensor plasmid.We measured OD600 and fluorescence of the strains at intervals.
Plasmid Construction(gene circuit)
BBa_K1649003 was cloned into pET28a using the NotI and XbaI sites. This plasmid contains a Kanamycin resistance gene and a pBR322 origin.

Strains
BL21 strains are used to harbor blue light sensor plasmid. The control is BL21 strain.
Culture
BL21 strains transformed with blue light sensor plasmid (marked as pCNGG) and BL21 strains ( negative control) were each picked 3 colonies and grown overnight (~12 hours) at 37℃ and shaken at 200 rpm in 3 ml LB containing antibiotic. The overnights are then diluted 1:200 into 2 mL fresh media containing antibiotics in 2 Costar 24-well plates. One plate was exposed with blue LED which we call p1 and the other were wrapped in aluminum foil and kept in darkness which we call p2. All plates were maintained at 37℃. Each strain was taken 100μl into a new, identical set of 96-well plates and then measured OD600 and fluorescence at intervals.

Result

Fig.1 fluorescence-time figure The figure shows a fluorescence level changing over time. The red line stands for the fluorescence in light, while the blue one in dark. We can see a great difference on level between two lines, also a more obvious downfall can be found on red line. These prove that our part does work.

Fig.2 od600-time figure The figure shows the cell growth in dark and light.

Source

kit plate