Creation of a cell-free chloroplast extract from Cannabis sativa and Nicotiana tabacum for biotechnological prototyping

Masterthesis

Beschreibung des Projekts – Project Description

Chloroplast biotechnology is mainly based on biolistic transformation and afterwards tedious regeneration of homoplasmic plants carrying transgenic chloroplasts. Unfortunately, this time-consuming creation of results easily takes months, creating a detrimental bottleneck during research purposes. Coupled transcription and translation in cell-free chloroplast extract could be a suitable way to shorten this process as a means of prototyping constructs for proper processing before biolistic transformation. Building upon the work of Clark et al. (unpublished) and the project of the Marburg 2021 iGEM project, a working cell-free chloroplast extract from Cannabis sativa and Nicotiana tabacum shall be created.

Aufgabenstellung – Task

During the thesis, you will be isolating chloroplasts using two different protocols. The fitness of the chloroplasts shall be compared between the two isolation protocols. Afterwards, the cell-free extract is created from the isolated chloroplasts and by means of NanoLuc luciferase assay tested for activity. In parallel, heterologous NanoLuc luciferase is produced and isolated from Nicotiana benthamiana as a control for the assay. Notable methods include Agrobacterium mediated gene transfer, Northern and Western Blotting, chloroplast isolation, in-vitro transcription and translation, etc. Vorkenntnisse – Previous knowledge Not necessary, but a profound interest in plant biotechnology and metabolic engineering is strongly encouraged! Previous knowledge in cell-free transcription and translation is advantageous.

Zeitraum – Timeframe

6 months, earliest starting date 3rd of November 2022. 9 months when the research internship shall be carried out in advance.

Arbeitsgruppe und Ansprechpartner

Kontakt über Prof. Heribert Warzecha, warzecha@bio.tu-darmstadt.de