This project proposed to investigate the feasibility of using DNase-SEQ to identify of regulatory elements in Chlamydomonas reinhardtii; with the view to producing a genetic toolkit for this alga.
We propose to run a pilot experiment to investigate the feasibility of using DNase-SEQ to identify of regulatory elements in Chlamydomonas reinhardtii; with the view to producing a genetic toolkit for this alga. DNase-SEQ is a powerful approach to identify transcription factor (TF) binding sites (He et al. 2014) which can then be utilised as genetic parts. To date there have been no reports of DNase-SEQ being applied to C. reinhardtii so the first stage of the project will be to establish the procedure.
As a test case we will focus on identifying regulatory elements that control the induction of the algal carbon concentrating mechanism (CCM). The reason for this is that the Griffiths lab is interested to understand the CCM, and a set of genetic parts that induce expression upon CCM induction could serve as useful tools for future analyses, such as high throughput screening of carbon concentrating components which could be engineered into higher plants for crop yield improvement.
Additionally, exploring DNase-SEQ data can be difficult for those without bioinformatics experience; we therefore aim to develop an open access, online tool to facilitate this process.
Ms Kher Xing (Cindy) Chan,
Graduate Student, Department of Plant Sciences, University of Cambridge
Dr Steven Burgess,
Postdoctoral Researcher, Department of Plant Sciences, University of Cambridge
Ms Marielle Vigouroux,
Computational biologist, John Innes Centre, Norwich
Genetic Parts for Algae
This project aims to run a pilot experiment to investigate the feasibility of using DNase-SEQ to identify the regulatory elements in Chlamydomonas reinhardtii, with a view to add to the existing toolkit for the alga. As a test case, we will focus on identifying regulatory elements which controls the induction of the algal carbon concentrating mechanism (CCM). The project is divided into three phases (Figure 1):
- Cell synchronisation and nuclei isolation.
- DNase I digestion and DNase-SEQ
- Bioinformatics and building of regulatory network
Phase I: Cell Synchronisation and Nuclei Isolation
Cell synchronisation was established using the method published by Mitchell et al. (2014) and cells can be synchronised and achieve the cell density required for nuclei isolation in a week.
Nuclei isolation was performed using protocol of Dr. Ivan Llorens but the quality and quantity of nuclei obtained was insufficient to continue with DNase I digestion. The nuclei were clumped together and degradation was observed (Figure 2). The outcome of this part of the project was presented as a poster during the OpenPlant Forum 2016 in Norwich.
The issue of clumped nuclei was addressed using the protocol with a nuclei isolation buffer of lower pH (Sikorskaite et al. 2013) (Figure 3). However, the optimal number of cells used for nuclei isolation needs to be determined in order to obtain sufficient amount of nuclei for DNase I digestion.
Phase II: DNase I Digestion and DNase-SEQ
This phase will be conducted in the first quarter of 2017 upon achieving the optimal conditions for nuclei isolation process.
Phase III: Bioinformatics and Building Regulatory Network
Marielle Vigouroux of JIC uses a practice dataset from Dr. Steven Burgess to build the regulatory network while waiting for the data from our project.
Due to commitment to other projects, the original schedule for this project is delayed but we would like to extend the current project to the first quarter of 2017. The final version of all protocols used for this project will be uploaded to protocols.io by the end of the project.
Follow On Plans
Sequencing is proposed to be done by end of March and the remaining funding from the initial £4000 will be spent on it. The additional £1000 will be spent on building the regulatory network and the website associated with it.