Part:BBa_K1884008
PSAD Promoter+GalAD-CRY2+UAS+PsbA+PSAD Terminator
The device we constructed this year is one of the functional plasmids in the light-mediated expression system,followed the example of yeast two-hybrid system. With BD-CIB1 which is constructed in the other device(BBa_K1884007), light-mediated expression system, followed the example of yeast two-hybrid system, will be activated and starting to transcript PsbA(BBa_K1884005) 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.
AD-CRY2(BBa_K1884002), a 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.
Upstream activating sequence(UAS)(BBa_K1884004), is a cis-acting regulatory sequence which is a region of non-coding DNA, regulating the transcription of nearby genes. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the enhancer in multicellular eukaryotes.
PsbA(BBa_K1884005), is a coding gene that can transcript into microRNA named by the protein its regulate.The protein which is regulated by this gene is D1 protein (also known as PsbA), which forms the reaction core of PSII as a heterodimer with the D2 protein. In higher plants, the N-terminal residues of both proteins, which are exposed to the stromal surface, can be reversibly phosphorylated.
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)
Usage
This device will work with BBa_K1884007 in our system, following photographs will show the level of hydrogen production when the light-mediate controlled yeast-two-hybrid system works.
Placing transgeosis green algae with 250ml TAP medium in blue light until the green algae grows to Exponential phase, we use gas chromatograph to measure the hydrogen production. Fig 2-3 will shows the result of hydrogen production.
We set up a control group that the green algae without our device in a isolate system,so that Fig 2 shows the proportion of diffrent gases in the air.There are three paek values in this photograph. The highest peak reperents the proportion of nitrogen and the peak in the middle reperents the proportion of oxygen.The lowest peak reperents the proportion of hydrogen.
Fig 3 shows that when the isolate system cultured the transgeosis green algae into Exponential phase, the peak value of hydrogen is higher than the control group which means that our system has been activated and prove the microRNA has targeted the PsbA gene coding D1 protein in photosynthesis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3123
Illegal NotI site found at 3221 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1560
Illegal BglII site found at 2019
Illegal BamHI site found at 2498
Illegal XhoI site found at 3052 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1444
Illegal AgeI site found at 2173 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1796
Illegal BsaI.rc site found at 1205
Illegal SapI.rc site found at 1313
Illegal SapI.rc site found at 3384
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