Difference between revisions of "Part:BBa K1884009"

Latest revision as of 02:04, 20 October 2016

PSAD Promoter+GalAD-CRY2+UAS+FNR(fumarate and nitrate reduction)+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 Ferredoxin-NADP(+)Reductase(BBa_K1884006), which is on the 3 prime end of Upstream activating sequence(BBa_K1884004) in green algae.

Biology

PASD promoter 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, 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) 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

Ferredoxin-NADP+ reductase (FNR) is the key enzyme catalyzing the reaction of electron transfer from ferredoxin (Fd) to NADP+, leading to production of NADPH mainly used for carbon dioxide (CO2) fixation in photosynthesis. Under anaerobic condition, FNR has been proposed to be one of the components competing for photosynthetic electrons from Fd with hydrogenases in Chlamydomonas ^[1]. Recent data from an in vitro experiment indicated that, under anaerobic condition supporting H2 production, there is a significant loss of photosynthetic electrons toward NADPH formation ^[2]. Using a proteomic approach, we have previously identified FNR as one of the differentially expressed proteins in Chlamydomonas that undergo sulfur-deprived H2 photoproduction process ^[3].

PSAD terminator 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 FNR gene coding Ferredoxin-NADP+ reductase in photosynthesis.

Sequence and Features