1 Green Biotechnologie - Protocol
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Zwecks der Planung: Vielleicht ist es möglich den Hauptimput bis vor Weihnachten im Wiki zu haben. Ich werd meinen Teil bis 16.12 reinstellen.
- Wenn erwünscht können wir uns ja vo Weihnachten nochmal treffen falls fragen usw. aufgetaucht sind.
- Nach den Ferien (bei mir ab 8.1. möglich) meeting mit uns 5 zur Vorbereitung für das meeting mit Prof. Obermeyer (zum Fragen klären).
- Selbiges dann möglichst schnell.
- Abgabe am besten in Zweiter Januarwoche.
Wichtig wär mir, dass wir nich noch in der Prüfungszeit dran sitzen. Wäre vielleicht auch vorteilhaft für die Note wenn wirs schnell abgeben. Wenn sich das jemand anders/schneller/o.ä. vorstellt, bitte reinkritzeln :)
University of Salzburg Department of Molecular Biology
Experimental course: „Green Biotechnology: molecular farming“
Teacher: Dr. Gerhard Obermeyer Tutor: Peter Lughofer, Bakk.-Biol.
Students: Doris Trapin (Mtr.-Nr. 0421277) Barbara Stöger, Bakk.-Biol. (Mtr.-Nr. 022085, St.-Kzl. 066832) Christoph Janig (Mtr.-Nr. 0520325) Cornelius Fischer (Mtr.-Nr.0521481) Cornelia Fischer (Mtr.-Nr. 0421029)
The aim of this course is to transform Nicotiana tabacum by the soil bacterium Agrobacteria tumefaciens (strain LBA 4404). The integration of T-DNA into the tobacco plant-genom is tested by different methods like histoGUS-assay, fluoGUS-assay and GFP-assay. The right gene-sequence is controlled by plasmid digestion and PCR and the protein content and quality is controlled by SDS-PAGE and Western-Blot.
Key words: Agrobacterium-mediated transformation, binary vector pBI121 and pBINPLUS.pImpect1.3T, ß-glucuronidase, GFP, Nicotiana tabacum, Italic text
In our course we trie to introduce a foreign gene encoding for a marker activity (ß-glucuronidase, GFP) into tabacco plants.
Agrobacterium tumefaciens is an soil bacterium which is able to infect wounded parts of plants to form galls growth by tumor-inducing plasmids, short Ti-plasmids. This Ti-plasmids contains the oncogenes, vir genes, T-DNA, the left (LB) and the right (RB) border. The T-DNA is the part which is transferd into the plant cell. In the course we work with two smaller, 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.
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. link title
Materials and Methodes
1.Transient expression of ß-glucuronidase and GFP in tobacco leaves
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.
For the histoGUS-experiment 16 leave parts were put into a 50 ml Grainer-tube including the histo-x-Gluc-solution. 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).
- · 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.
- · 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.
- ·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.
2. GUS assay in leaves and leav homogenates
Fluorometric GUS assay
Stuff to work with
4. Isolation and digestion of plasmids and PCR
Material and methods
Isolation of plasmid
To verify that our two agrobacs cultures*, with which the transformation had been done, de facto were containing the respectively Ti-plasmid-construct, we had to isolate the plasmid for further treatment. The plasmid isolation was done by a Wizard Plus SV Minipreps DNA Purification System from Promega. As the protocol was originally designed for E.coli, we had to modify it a little to make it suitable for A. tumefaciens (e.g.the addition of lysozyme for the cell wall digestion).
The determination of if and how well the plasmid isolation had worked was done by a photometric assay (at a of 260 and 280 nm) and an agarose gel (1% w/v + EtBr in TAE buffer).
Plasmid digestion and PCR
The double digestion of the pBI121 was done by the enzymews BamH1 and SacI. The double digestion of pBINPLUS.pImpact1.3T was done by PacI and AscI. Also a single digestion by PacI was done. This enzymes were used as they should cut the plasmid only once, yielding to our gene of interest (GUS and GFP).
For the PCR the primer pair MS18_GUS_fwd and MS19_GUS_rv were used for the pBI121 and the primer pair MS11_PrbcS1_fwd and MS10_TrbcS1_rev were used for the pBINPLUS.pImpact1.3T., yielding to the amplification of our gene of interest. The Taq-polymerase and its buffer were obtained from Stratagene.
The DNA products of digestion and PCR and also intact plasmids (as control) were determined on an agarose gel(1% w/v + EtBr in TAE buffer).
(*2 agrobacs cultures: agrobacterium strain LBA 4404 with pBI121 and pBINPLUS.pImpact1.3T grown on a YM medium supplemented with antibiotics: 100 µg ml-1 streptomycin, 50 µg ml-1 kanamycin)
Results of plasmid digestion and PCR
Gelphoto kommt hierher - christoph bitte hier einfügen
Results of plasmid digestion
In the first column we see the ladder of the bp-size-marker.It's not an optimal one, because our plasmids are around 15.000 bp and the greatest kb-marker has 10.000 bp. To the second column was applied the undigested pBINPLUS.pImpact1.3T (as a control), which is composed of 15.168 bp and should have a supercoiled form. In the gel we can see a very dark thick band, which is situated -as expected - over the 10.000 bp-marker. To the third column was applied the pBINPLUS.pImpact1.3T having been incubated with one enzyme. What we can see are 2 bands: the lower one must be the supercoiled form of the undigested plasmid, like in column 2, auch wenn they aren't at the same high. The upper one must be the linearized plasmid (through a unique dsDNA-cut by the enzyme. To the fourth column was applied the pBINPLUS.pImpact1.3T having been incubated with two enzymes. The digestion should lead to 2 fragments (bands): the cutted-out GFP(with 2.840 bp) and the rest-plasmid (15.168 bp - 2.840 bp = 12.328 bp). But on the gel we can see 5 bands; this means that the enzymes didn't work so specifically, which has led to three addidional bands. The uppest band at ahigh of ~ 12.000 bp could be the (expected) rest-plasmid, as it is situated a bit lower than the undigested supercoild form (column 2 and 3). The second, third and fifth band (seen from up to down) must be unspecific bands. The remaining fourth band at a high between 2500 and 3000 must be the expected cutted-out GFP.
To the sixth column was applied the undigested pBI121 (as a control), which is composed of 14.758 bp and should have a supercoiled form. In the gel we can see 2 bright bands, both over the 10.000 bp-marker: one must be the expected supercoiled undigested plasmid DNA (perhaps the upper on), the other (lower)one could be a great fragment of it. This is unexpected and strange,as we hadn't added any enzymes to the plasmid. To the seventh column was applied pBI121 having been incubated with two enzymes. The digestion should lead to 2 fragments (bands): the cutted-out GUS (with 1.893 bp) and the rest-plasmid (14.758 - 1.893= 12.865 bp). In this case, we see only the 2 expected bands at the corresponding high (without any unspecific digestion products/fragments).The rest-plasmid is bright and -with a bit of imagination- a very bright (expected) GUS-band can be seen.
Results of PCR
To the nineth column was applied a sample-aliquot of the PCR-reaction for the GUS-gene (of the pBI121). The expected PCR-product should have 1812 bp. Unfortunately, we don't see anything in the gel. This means that there must be happened an error, "wahrscheinlich" in pipetting the primer or the Paq-polymerase. To the tenth and eleventh column was applied a sample-aliquot of the PCR-reaction for the GFP-gene (of the pBINPLUS.pImpact1.3T ). The expected PCR-product should have 1137 bp. In this case the expected band can be seen an the corresponding high (beween the 1000 and 1500 bp-marker). In the tenth column there can be seen also another band at a high that "equals/corresponds to" the upper band of column 6, which we suppose to be the undigested supercoiled form of the pBINPLUS.pImpact1.3T.
The enzyme GUS cleaves the Indigo rest of the substance x-Gluc (Indigoglucuronicacid). The indigo will show a blue colour only in cells that had been infiltrated by GUS. Bluestained leaves were documented by a camera. As we can see on the pictures above there is an increase of the staining with number of days. On day 0 no blue coloured areas were visible because a longer time of incubation is needed. We got the best results on day 4 where the highest amount of gene-expression by ß-glucuronidase is visible. The results from samples of day 5, 6, and 7 may be declined because the leave parts dried out in the incubation chamber.