Molpharming

Green Biotechnologie - Protocol
'''This page would give us the posibility to create the protocol for the course Green Biotechnologie interactiv. And makes it easier than sending word documents around the world ;)'''

Click edit (right) to edit text below the corresponding headline or click edit (top) to edit this page. Click discussion (top) to make a comments. Every change will be logged in the history (top).

Here you find how to format text: http://de.wikipedia.org/wiki/Hilfe:Textgestaltung

Here you find a general introduction in WIKIA: http://www.wikia.com/wiki/Help:Tutorial

Log in and create an username!

Add this page to your Bookmarks to reach it quickly.

Greetings Coni

University of Salzburg Department of Molecular Biology

Experimental course: „Green Biotechnology: molecular farming“

Nr. 437.711

WS 2007/08

Teacher: Dr. Gerhard Obermeyer Tutor: Peter Lughofer, Bakk.-Biol.

Students: Doris Trapin (Mtr.-Nr. 0421277) Barbara Stöger (Mtr.-Nr. 022085, St.-Kzl. 066832) Christoph Janig (Mtr.-Nr. 0520325) Cornelius Fischer (Mtr.-Nr.0521481) Cornelia Fischer (Mtr.-Nr. 0421029)

1. Introduction
The aim of this course is to transform Nicotiana tabacum by the soil bacterium Agrobacteria tumifaciens (strain LBA 4404). The integration of T-DNA into the tobacco plant-genom is tested by different methodes like histoGUS-assay, fluoGUS-assay and GFP-assay. The right gen-sequence is controlled by PCR and the protein content and quality is controlled by SDS-PAGE.

Agrobacteria tumifaciens have a Ti-plasmid witch contains the oncogenes, vir genes, T-DNA, the left (LB) and the right (RB) boarder. In the course we work with two smaler, so called “disarmed” Ti-plasmids. We use the first binary vector pBI121 for the GUS-assays. There the T-DNA region (6193bp) contains RB, expression cassettes for an neomycin phosphotransferase II (NPTII) selection marker and a ß-glucuronidase (GUS) reporter gene, and the LB. (Chen at al 2003) The second Agrobacteria culture, with the pBINPLUS.pImpact1.3T:sGFP vector, containing a GFP-, a His-Tag and a KDEL region, is used for the GFP-assay.

The transforming strategy contains three major steps, cloning the foreign cDNA sequence into the binary Ti vector, introduction of the Ti vector into Agrobacterium and the transformation and regeneration of the transgenic plants.

2.	Transient expression of ß-glucuronidase and GFP in tobacco leaves
Introduction

Nicotiana tabacum-plants (whole leaves and leave disks) were transformed by agro-infiltration with disarmed Ti-plasmids of Agrobacterium tumifaciens strain LBA 4404. For the GUS assay we used an agrobacs strain containing the vector pBI121 with the beta-glucuronidase-gen. For the GFP assay the Agrobacs strain contained the pBINPLUS.pImpact1.3T vector with the green fluorescence protein. GFP and GUS can be detected by different methods which are described later. For the negative control the plant material were treated the same way, but in the absence of agrobacs. And for positive control stable plant material was used and treated in the same way, with agrobacs.

Material and Methodes

Agro-infiltration protocol

Our Tutor, Peter Lughofer, prepared the 2 agrobacs cultures: per culture 10 ml of Agrobacterium (LBA 4404 with pBI121 and pBINPLUS.pImpact1.3T grown in YM medium supplemented with antibiotics: 100 µg ml-1 streptomycin, 50 µg ml-1 kanamycin) to inoculate an 90 ml YM medium. For vir gene induction he added 10 mM Mes (ca. pH 5.6) to acidify the medium. The culture were grown over night (28°C) with 200 µM acetosyringone (this signal substance activates the agrobacs to conquere an injured plant). The bacteria were pelleted at room temperature, ca. 4000 xg, and resupend in water to a final volume of 50ml (measured OD for pBI121: 0.542, OD for pBINPLUS.pImpact1.3T: 0.518). The agrobacs were incubated for 1 h at RT meanwhile 18 leave parts and 54 leave disks from Nicotiana tabacum were cut out.

Vacuum infiltration

For the histoGUS-experiment 16 leave parts were put into a 50 ml Grainer-tube including the bacterial (pBI121) suspension. The same procedure for the fluoGUS-experiment, here we used 24 leave disks and also the bacterial pBI121 suspension. For the GFP we also used 24 leave disks and the pBINPLUS.pImpact1.3T bacterial suspension. It is important that all the leave material is covered by the suspension.

These Grainer-tubes were placed, without the cap, into the excicator and apply a low pressure of 60-80 mbar for 20 min. The infiltrates leaves, for the histoGUS-assay, were put into 8 big petri dishes (each two leave parts). The leave disks for the fluoGUS-, and GFP-assay were put into 16 small petri dishes (each three leave disks). Shortly after they were incubated at 24°C. We also got positive controls from stable transformed tobacco plants.
 * · Samples of leave parts were taken every day (day 0-7), for negative and positive control only one time. The GUS infiltration is tested by the GUS-assay at chapter 3.
 * · Samples of leave disks were taken every day (day 0-7), ), for negative and positive control each only one time. They were frozen at –20°C. With these samples the fluoGUS-assay and the GFP-assay was performed on day 7, see chapter 4 and 5.

Stuff to work with
Sketch by Mr. Obermeyer

enjoy;) The calibration curve of MU Foto HistoGUS Day1

Calibration
Callibration with Gain 60 Callibration with Gain 30 Callibration with Gain 45