Part:BBa_K1884001

Gal4BD-CIB1

Cryptochrome-interacting basic-helix-loop-helix 1(CIB1) is a protein-coded gene. The product of this gene expression is a basic helix-loop-helix (bHLH) protein which would interact with cryptochrome 2 (CRY2), a blue light stimulated photoreceptor, when exposed to blue light. This part is a Gal4 DNA binding domain fused to C terminus of CIB1.

Usage and Biology

This fusion protein is for use in a yeast-two-hybrid system,and a Gal4 DNA binding domian fused to its C terminus. In order to control DNA transcription by blue light, the system is based on a two-hybrid interaction in which a light-mediated protein brings together two halves of a split transcription factor. CRY2 will disconnected with CIB1 in the dark and halt the DNA transcription.(Fig 1)

Figure 1. The diagram of light-mediate controlled yeast-two-hybrid system.

BD-CIB1 is 1533bp in length. Fig 2 shows the DNA sequence of BD-CIB1 is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of BD-CIB1 PCR product is rather high compared with DNA Marker, which indicates that the PCR product of BD-CIB1 is in a high concerntration.

Figure 2 The electrophoretogram of BD-CIB1 PCR product.

For confirming the gene of our fusion protien gene has been transformate into green algae, we extracted genome DNA of green algae and designed two pairs of primers in order to amplify BD and CIB1 respectively. Fig 3-4 shows the DNA sequence of BD and CIB1 are successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of BD and CIB1 PCR product is rather high compared with DNA Marker, which indicates that the PCR product of BD and CIB1 are in a high concentration.

Figure 3 The electrophoretogram of BD PCR product.

Figure 4. The electrophoretogram of CIB1 PCR product.

Improvement

This year the gene which code BD protein is improved from a part of basic part named (BBa_K1592005). The difference in our two systems lies in the two fusion proteins in the system.We combine AD with CRY2 as a fusion protein,combine BD with CIB1 as another fusion protein.And the two fusion proteins from HUST-China team 2015 project are AD-CIB1 and BD-CRY2.We regard this difference as an improvement because there are lots of AD’s restriction site in CIB1.So,if we want to get a complete AD-CIB1 after enzyme digestion,we have to do a lot of point mutation for CIB1.In theory, however, AD combined with CIB1 or CRY2 has no effect on the experimental results.Therefore, we believe that the combination of AD and CRY2 in constructing plasmids is a better choice for both project and biobrick experiments.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 218
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 637
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 137