Part:BBa_K1884007

PSAD Promoter+BD-CIB1+PSAD Terminator

This decive we constructed this year is one of the functional composite parts in the light-mediated expression system, followed the example of yeast-two-hybrid system. With AD-CRY2 which is constructed in other two device(BBa_K1884008 or BBa_K1884009),the light-mediate-controlled yeast-two-hybrid system will be activated and start to transcript the hydrogen producted genes(BBa_K1884005 or BBa_K1884006) which is on the 3 prime end of Upstream activating sequence(BBa_K1884004) in green algae.

Biology

PASD promoter(BBa_K1547005) is a plant promoter from the green algae Chlamydomonas reinhardtii. It is a high expression promoter that encodes for a ferrodoxin-binding protein of photosystem I.This year we decided to built the light-mediate-controlled system in green algae Chlamydomonas reinhardtii.Based on the origin of PSAD promoter, we thought it would be fully competible in our project.

BD-CIB1(BBa_K1884001) 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.

PSAD terminator(BBa_K509003) is a plant specific Terminator, and serves to increase expression of a gene placed upstream. It works in collaboration with PSAD promoter(BBa_K1547005). Both of these parts are activated by sunlight.(Fig 1)

Figure 1. The diagram of this composite part in pSB1C3 Backbone.

Usage

This device will work with BBa_K1884008 or BBa_K1884009 in our system, following photographs will show the level of hydrogen production when the light-mediate controlled yeast-two-hybrid system works.

Gas chromatography is an accurate quantitative measurement to measure the volume of every gas in the atmosphere. Puting our transgenosis green algae under the light that lack of blue light(Normally we use red light. wave length:660nm, halfwidth:17.9nm, main wave length:542.6nm, excitation purity:0,989). When the Exponential phase comes to our green algae, Blue light is for use in guiding the expression of microRNA-D1, and increasing the output of hydrogen production.

For confirming our light-mediate controlled yeast-two-hybrid system would/ work, the repoter we use is that to calculate the volume of hydrongen production. First, transgenosis green algae has been regulated by blue light for 4 and 8 hours, then we put them back to red light to recover for 16hours after we finish measuring the production of hydrogen. After that, we sitll use blue light to regulate the expression for 4hours、8hous and 12hours, measuring the level of hygrongen, as a result of which, the green algae which are in blue light have the higher level of hydrogen production and they would recover constantly in red light. Based on this result, we know that our light -mediate congtrolled yseat-two-hybrid system is fully functional to work, besides, we can regulate it more than once in order to produce hygrogen constantly. the following photographs will show the results of gas chromatograph.(Fig 2-8)

Figure 2. transgenosis green algae in blue light for 0 hour at the first day.

Figure 3. transgenosis green algae in blue light for 4 hours at the first day.

Figure 4. transgenosis green algae in blue light for 8 hours at the first day.

Figure 5. transgenosis green algae in blue light for 0 hour at the second day.

Figure 6. transgenosis green algae in blue light for 4 hours at the second day/b>.

Figure 7. transgenosis green algae in blue light for 8 hours at the second day.

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Figure 8. transgenosis green algae in blue light for 12 hours at the second day.

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 1118
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1537
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1037
  Illegal SapI.rc site found at 2475