Construction of knock-out vectors for nifD and vnfDG in Nostoc 210A, 232 and 213 and N6

• Main Author: Borgmann, Martin Josef, 1991-
• Other Authors: Háskóli Íslands
• Format: Report
• Language: English
• Published: 2015
• Subjects: Líffræði; Arctic; Subarctic
• Online Access: http://hdl.handle.net/1946/23470

This is the report of a seven weeks participation in the project Alternative nitrogenases in cyanobacteria of Arctic and Subarctic ecosystems which is supervised by Ólafur S. Andrésson. The aim of the project is to investigate what drives the selection of the alternate Vnf nitrogenase under certain conditions in cyanobacteria in lichens, mosses and streams. Another objective is to characterize the nitrogenase activities and Vnf clusters i.e. gene expression, acetylene reduction, discrimination of the isotopes 15N and 13C under different conditions of Molybdenum (Mo) and Vanadium (V) availability, temperature and light. Also it is being evaluated how much the Vnf nitrogenase is involved in the nitrogen budget of subarctic and arctic ecosystems. It is assumed that ∆15N values can be used as a proxy of V nitrogenase contribution. Another aim is to characterize the Mo and V nitrogenase and their regulation in sequenced cyanobacterial strains (N210A, N213, N232 and N6). Knock out (KO) constructions will be utilized for this. They are made by inserting antibiotic resistance cassettes into key genes of each system. nifD is a gene transcribed in the presence of molybdenum and vnfDG is transcribed in the absence of molybdenum. During the seven weeks course Research project in biology for foreign students (LÍF039L) essential pieces for the KO vectors have been constructed by PCR amplification of sequences flanking the insertion points of nifD and vnfDG. The PCR constructs also have adaptor sequences for facile a vector construction using the Gibson assembly procedure. [1, 2, 3] This report will explain what has been done and for what reason and finally sum up the results.