As we mentioned before that the two step cultivaton has some defects, so we want to use other methods to control photosynthesis instead of sulfur deficiency. So we come up the idea of using miRNA to control it.
miRNA is a type of noncoding RNA which can regulate the cell function by inhibiting the expression of target gene. So we design the artificial miRNA especially targeting the key protein D1 in PS II 。When we induce Chlamydomonas cell to overexpress this miRNA, the expression of D1 protein will be inhibited which will decrease the photosynthesis. So that the oxygen level will be reduced and the hydrogenase will be activated and will be able to produce hydrogen as we wish. This method can replace the two-stage cultivation perfectly and has a higher efficiency.
light-mediated expression system
We plan to use a inducible factors--illumination to control miRNA to produce hydrogen. In fact, the light-mediated expression system have already been built inside animal, bacteria, yeast, but not in green algae yet. Building an efficient, easy-controlled extraneous light-mediated system for Chlamydomonas is pretty essential and also a great breakthrough. Therefore we decided to use light with different wavelength(blue light and white light which filtered blue lights) to control the hydrogen production of the Chlamydomonas.
Followed the example of yeast two-hybrid system, we had built a light-mediated expression system which apply to the H2-production in green algae C. reinhardtti. Specifically, the system is based on 2 main parts. The first one is light-induced transcriptional control which depends on a blue light stimulated photoreceptor, cryptochrome 2, CRY2, and its interacting partner CIB1. CIB1 and CRY2 are 2 parts, combining with each other under blue light. The second part is a two-hybrid interaction , in which a light-mediated protein interaction brings together two halves, a binding domain, BD, and an activation domain , AD. Binding domain can specifically recognize and combine with UAS domain. And activation domain can activate the function of UAS when combining it. For our project, we link BD with CIB1 and AD with CRY2. What’s more, UAS is a kind of unique inducible promoter, only promoting down stream gene when combining with AD.And our down stream gene can translate miRNA, which can regulate the expression of D1 protein.
When there is a blue light, CIBI will combine with CRY2, which will bring the AD to UAS and will start the whole chain of gene translation so that our microRNA is produced. So the D1 protein can be inhibited and we can keep on producing hydrogen. After a period of time we will stop the blue lights and change into white lights without blue wavelength. so that the production of microRNA will be stopped and the photosynthesis will start again. In conclusion, when under the blue light the microRNA can be produced so as the hydrogen. And when under the white light there will be no hydrogen being produced but photosynthesis gets to continue. We can control algal cell produce hydrogen continuously by Intermittent illumination between blue light and white light.