Part:BBa_K1884002

VP16AD-CRY2

Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact with CIB1. The CRY2/CIB1 interaction do not need extra exogenous cofactors. The binding will reverses in minutes in the dark. This part is a VP16 DNA activating domain fused to N terminus of CRY2.

Usage and Biology

This fusion protein is for use in a yeast-two-hybrid system, and a VP16 DNA activating domian fused to its N 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.

To make this parts RFC 10 competible, we use Site-directed mutagenesis to mutate the old sequence so that none of these four Restriction enzyme(EcoR1/Xba1/Sep1/Pst1) can digest this gene.（Fig 2）

Figure 2. the location of the mutated basic group.

AD-CRY2 is 2106bp in length. Fig 3 shows the DNA sequence of AD-CRY2 is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of AD-CRY2 PCR product is rather high compared with DNA Marker, which indicates that the PCR product of AD-CRY2 is in a high concerntration.

Figure 3. The electronphoretogram of AD-CRY2 PCR product.

After transmate the plasmid into green algae, we extract genome DNA of green algae. Fig 4-5 shows the DNA sequence of AD-CRY2 is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of AD-CRY2 PCR product is rather high compared with DNA Marker, which indicates that the PCR product of AD-CRY2 is in a high concentration.

Figure 4. The electronphoretogram of AD PCR product.

In this photograph(Fig 4), the length of AD is 475bp. because in our project we use AD-UAS instead of AD in order to built our system easily. For making a functional light controlled yeast-two-hybrid system, we separate AD from AD-UAS and fused it with CRY2.

Improvement

This year the gene which code AD protein is improved from a basic part named (BBa_K105001). the most important improvement of our gene is that we optimize codon in this gene in order to transcript in green algae stably.(Fig 5)

Figure 5. The location of optimized codon in Activated domain.

The difference between (BBa_K1592006) from HUST-China and this part is that we combine AD with CRY2 as a fusion protein instead of AD with CIB1. 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 BglII site found at 660
  Illegal BglII site found at 1119
  Illegal BamHI site found at 1598
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 544
  Illegal AgeI site found at 1273
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 896
  Illegal BsaI.rc site found at 305
  Illegal SapI.rc site found at 413