Part:BBa_K3285000

Blue Light Repressible Promoter

This composite promoter consists of 6 basic parts that include 2 promoters, 3 coding sequences and one double terminator(Fig.1)

Fig.1: The basic gene circuit of the blue light repressible promoter

USAGE AND BIOLOGY

As we can see in Fig.2, there is a constitutive promoter that constitutively expresses the yf1 and fixJ genes. YF1 is a fusion protein made of YtvA (Light sensitive protein) and FixL (phosphorylates FixJ). The phosphorylated FixJ induces pFixK2 promoter and hence mrfp is expressed. As a result the RFP is constitutively expressed when there is no blue light (Fig.2)

Fig.2: The working of the promoter in the absence of blue light

Now, in the presence of blue light, the conformation of Ytv1 changes hindering the phosphorylation of FixJ by FixL leading to a reduced expression of mrfp gene. Increasing the intensity of blue light further hinders this interaction leading to an even lesser expression of mrfp gene. As a result, with increasing intensity of blue light, RFP production reduces.

Fig.3: The working of the promoter in the presence of blue light

We want to use this promoter to regulate the level of mutD5 (codes for the faulty epsilon subunit of DNA polymerase III of bacteria) in the cell thereby resulting in different levels of mutation rates in the bacteria. Our project aims at developing a tool for attaining tunability of mutation rates in E.coli using an external physical inducer (such as blue light) which will help in performing directed evolution faster and more easily.

CONSTRUCTION

Restriction free cloning (PCRs) was used for obtaining the final composite promoter and all other intermediate parts which will be described below. First, Dterm, pFixK2 and mrfp were ampilfied from their respective plasmids. For amplification of parts from plasmids, 50-100 ng of template was used. For Dterm, annealing temperature (A.T) was 53 deg., for pFixK2 the A.T was 58 deg. The extension period (E.P) for both was 18 sec. For mrfp, A.T was 54 deg. and an E.P of 50 sec was used (Fig.4) The resulting bright bands obtained in each case were gel extracted and purified.

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
  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
  Illegal AgeI site found at 2815
  Illegal AgeI site found at 2927
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

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