Day Working Plan S1 instructions, Introduction Isolation of genomic DNA from Populus tremula

Transkript

1 Date Day Working Plan S1 instructions, Introduction Isolation of genomic DNA from Populus tremula Gel analysis of DNA-preparation 2. PCR for the amplification of pre-mirna and target gene PtMADS8 on genomic DNA from Populus tremula Preparation of competent E.coli cells Purification of PCR-products using gelextraction Restriction digests of the vectors and PCR products 2. Purification of digested vectors and PCR products Ligation of the vectors with the PCR products 2. Preparation of the LB plates 3. Transformation of the ligations into competent E. coli cells Evaluation of Transformation & set up of liquid E. coli cultures of the transformed cells 2. Bioinformatics part I (Lydia Gramzow) Small-scale preparation of plasmid DNA from transformed E. coli 2. Restriction digest to control for the insertion of the PCR products 3. Preparation of sequencing of plasmid DNA for PtMADS8 & premirna Preparation of YEB plates 2. Transformation of Agrobacteria GV Transformation of Nicotiana benthamiana leaves 9. bis 12 Visualisation of the GFP signal using a microscope Evaluation of Sequencing (Lydia Gramzow) - Day Bioinformatics part III (Lydia Gramzow) Examination (Testat) and hand over of protocols

2 WS 2013/2014 Practical Course mirna & regulation of gene expression in plants RNAs global regulators of gene expression in eukaryots. Several types of very short RNAs repress (or silence) expression of genes with homology to those short RNAs. These small RNAs are named different according to their origin. MiRNAs for example are produced from precursor RNAs which are encoded by genes expressed in cells. Figure 1: RNAi silencing. RNAi switches off the expression of a gene when dsrna molecules that have homology to that gene are introduced, or made, in the cell. This effect involves processing of the dsrna to make sirnas and mirnas by the enzyme Dicer. The sirnas and mirnas direct a complex called RISC (RNAinduced silencing complex) to repress genes in three ways. It attacks and digests mrna that has homology with the sirna; it interferes with translation of those mrnas; or it directs chromatin modifying enzymes to the promoters that direct expression of those mrnas. (Adapted, from Hannon G.J Nature 418: , Fig. 5) Watson Molecular Biology of the Gene Many plant micrornas down-regulate targets coding for transcription factors. Thus, micrornas represent master regulators in developmental processes, growth, and environmental interactions. The functional micrornas mature micrornas are usually very short sequences and comprise about 20-25nt. This fragment is excised from a long and variable transcript, a precursor which is characterized by a stem-loop shape. A maturation of micrornas comprises three fundamental intermediate stages (pri-microrna, pre-microrna, and mature microrna) and involves a broad range of proteins (figure 2). Most microrna genes are located within intergenic regions and are mostly transcribed by Polymerase II. The primary transcript is denoted as pri-microrna. In addition to polyadenylation and 5 capping, the pri-microrna can undergo a canonical splicing process. Due to the extended regions of complementarity the pri-microrna already exhibits an imperfect stem-loop structure based on intramolecular hydrogen bonds. The unpaired 5 and 3 ends of the pri-microrna fold-

3 back structure are trimmed. This process is localized in the nucleus and is mediated by a multiprotein complex of DICER-LIKE proteins (DCL), SERRATE (SE), HYPONASTIC LEAVES1 (HYL1), and cap-binding protein complex (CBC). Besides the existence of four DICER-LIKE proteins in plants, only DCL1 and DCL4 are assumed to be involved in the microrna maturation. Most micrornas are processed by DCL1, whereas DCL4 is supposed to recognize extended stems in the hairpins of evolutionary young micrornas. This step leads to the premicrorna sequence. Figure 2: Maturation of a mirna. The functional microrna emerges from the corresponding locus by passing through multiple intermediates. The genomic sequence is transcribed and processed into the pri*-microrna which is further metabolized via a pri-microrna and pre-microrna to the mir:mir* duplex. One of these two strands is getting included into the RNA induced silencing complex (RISC) as the mature sequence. Modified after Bartel, The pre-microrna structure is further processed by a second excision, also mediated by DCL. A double strand complex of the mature microrna and the complementary part (mir*) is generated where both fragments show a 2nt overhang at the 3 end. The binding of both strands in this duplex is near-perfect. Usually, less than 4 mismatches and no gaps or loops are present. The length of the resulting mir:mir* duplex is DCL dependent. The cleavage positions of DCL are determined by the structure of the pre-microrna. The two strands of the mir:mir* duplex are methylated on the 3 ends by HUA Enhancer1 (HEN1), to protect the duplex from degradation. The mir:mir* duplex is exported by HASTY (HST) into the cytoplasm. One strand of the methylated mir:mir* duplex is integrated into a large protein complex, referred to as RNA induced silencing complex (RISC). This incorporated part of the duplex defines the mature microrna. The term microrna refers to this about 21nt mature section only, not regarding the precursor stages. By acting as the guide strand, through its complementarity to the target mrna, the mature sequence mediates the specificity to the target. The anti-sense strand

4 (mir*) is degraded after separation. Besides the mature microrna sequence, the basic compound of the RISC is a member of the ARGONAUTE protein family (AGO). ARGONAUTE mediates the cleavage in the middle of the target mrna, usually at position 10 to 12 of the microrna binding, but also performs a silencing via translational inhibition. Despite the well studied mechanism of action, mirnas and its target still are hard to determine. The aim of this practical course is to verify an interaction of a predicted mirna (using SplamiR) with an AGL17-like gene (MADS-domain transcription factor) from Populus tremula. We will try to verify this interaction using a GFP-fushionprotein assay in N. benthamiana (Figure 3). For this assay the genes for the mirna and the target gene will be amplified using PCR. The gene for the target gene will be cloned in plant-vector containing a GFP-Gen, so that a fushion protein will expressed after transformation of a N. benthamiana leaf with the plasmid (Figure 3 left part). Functional expression of the fusion protein will result in a green color after transformation. The gene for the premirna will also be cloned in a plant-vector and in a second experiment co-transformed with GFP-target gene-plasmid. An RNA-interference will be detected by repression of GFP-expression resulting in a red color (color of chlorophyll while activated by UV-light (Figure 3 rigth part). Figure 3: In planta assay Verification of mirna & target gene interaction using a GFP-fushionprotein assay in N. benthamina.

5 Figure 4: Methotical schedule for practical course Basis for practical course (please read carefully) Paper 1)

6 Paper 2) Paper 3) Protokoll Am Ende des Praktikums ( ) soll von jeder Praktikumsgruppe ein Protokoll zu diesem Praktikum abgegeben werden. Dieses sollte folgendermaßen aufgebaut sein: 1. Name, Datum, Gruppe, 2. Studiengang, Matrikelnummer 3. Kurze Einleitung (5-10 Sätze) zu den einzelnen Versuchsteilen 4. Material und Methoden (nur falls abweichend vom Skript!!) 5. Ergebnisse (Messwerte in Form von Tabellen; Auswertung, mathematisch, graphisch; Abbildungen selbsterklärend beschriftet) 6. Diskussion (Bewertung der Ergebnisse, Vergleich mit Literaturdaten (sofern vorhanden) bzw. mit zu erwartenden Ergebnissen, Fehlerbetrachtung) 7. Zur besseren Nachvollziehbarkeit sind Einleitung, Material und Methoden, Ergebnisse und Diskussion sind jeweils als Block zu den zu den einzelnen Versuchsteilen schreiben! (Die jeweiligen Blöcke sind auf der Übersichtsseite farbig markiert). Für den Bioinformatischen Teil (Dr. Lydia Gramzow) gibt es ein eigenes Skript.

7 Day 2 Isolation of genomic DNA from Populus tremula (Protocol Fang et al. 1992; Biotechniques) - Grind 1 plant leaf using liquid nitrogen - Transfer the tissue powder to a 2 ml tube - Add 600 µl extraction buffer and 50 µg RNase A (2.5 µl of a 20 mg/ml stock), mix by inversion - Incubate at 65 C for 20 min (Thermomixer) for the lysis of the cells - add 200 µl 5M KAc - mix by inverting - incubate on ice for 5 min - spin at x g for 10 min 4 C - transfer the supernatant to a new 1.5 ml tube. Avoid to transfer any particulate material - determine the volume of your sample - add 1 volume of isopropanol and mix thoroughly by inverting - incubate on ice for 20 min - spin at x g for 10 min 4 C - check for the pellet - gently remove all of the supernatant by pipetting - air dry pellet for 10 min - add 200 µl TE buffer and incubate at 65 C for 15 min - spin at x g for 5 min 4 C - transfer the supernatant to a new 1.5 ml tube and determine the volume of your sample - add 0.1 volume of NaAc and 1 volume of isopropanol, mix by inverting - incubate on ice for 10 min - spin at x g for 10 min 4 C - gently remove all of the supernatant by pipetting - add 500 µl of 80% EtOH, mix by inverting - spin at x g for 1 min 4 C - gently remove all of the supernatant by pipetting - air dry pellet for 10 min - add 30 µl of TE buffer and mix well by flicking the eppi The Extraction Buffer consisted of 100 mm Tris-HCl ph 8.0, 50 mm EDTA, 500 mm NaCl and 1% PVP Additional solutions are 5 M potassium acetate (ph 5.5), 3 M sodium acetate, isopropanol, and 80 % ethanol.

8 Fragen: 1) Berechnen Sie die Einwaage und benötigten Lösungen für die Herstellung des Extraction Buffers (200 ml). 2) Finale Konzentration Stammlösung Benötige Menge mit Einheit 100 mm Tris-HCl 1M 50 mm EDTA 500 mm 500 mm NaCl 5 M 1% (w/v)pvp Pulver zum Einwiegen 3) Was ist PVP und wofür wird es benötigt? Day 3 3. Bestimmung der DNA-Konzentration & Analyse der DNA auf einem Agarosegel DNA Konzentrationsbestimmung - 1 µl DNA zusammen mit 49 µl ddh 2 O in ein 1,5 ml tube geben - Gut mischen und alles in eine Küvette pipettieren - Photometrische Konzentrationsbestimmung mit ddh 2 O als blank - Ggf eine neue geeignete Verdünnung der DNA herstellen und erneut messen 2 Proben der DNA werden auf ein 1% Agarosegel aufgetragen DNA 500 ng DNA 100 ng ddh 2 O auf 8 µl ddh 2 O auf 8 µl 2 µl loading dye 2 µl loading dye - Ggf. eine geeignete Verdünnung der DNA herstellen (keine Volumina <1 µl pipettieren) - 5 µl Marker neben die Proben auftragen - Gel bei 100 V ca 1 h laufen lassen Fragen: 1) Welche Banden erwarten Sie auf dem Gel zu sehen? 2) Wie funktioniert die DNA-Konzentrationsbestimmung am Photometer? Was sagen die Werte 230/260 und 260/280 über die DNA-Lösung aus?

9 4. PCR für die Amplifizierung der pre-mirna und des Zielgens PtMADS8 auf genomischer DNA von Populus tremula Zur Klonierung eines Gens, wird in einen ersten Schritt das Gen mittels spezifischer Primer amplifiziert. Diese Primer enthalten zusätzlich an ihrem 5 Ende Erkennungssequenzen für die benötigten Restriktionsenzyme. Für das Zielgen sollen 2 verschiedene Konstrukte kloniert werden. Gruppe 1 bis 4 soll das Gen von Exon 2 bis zum Stop amlifizieren. Gruppe 5 bis 10 das Gen von Exon 4 bis zum Stop amlifizieren. Exon-Intron-Struktur : Figure 5: Exon-Intron-Structure of PtMADS8. Size of 1kb is indicated as a black line above the figure. Exons are shown in boxes, Introns as lines. Primer: For PtMADS8 amplification: PtMADS8_fwd_SmaI (Exon 2 oder Exon 4) and PtMADS8_rev_BamHI 10x Pfu buffer + MgSO 4 1x 10 mm dntp mix 200 µm primer forward 5 µm 0.5 µm primer reverse 5 µm 0.5 µm Genomic DNA (100 ng) µl Pfu DNA Polymerase 0.5 µl PCR water up to 50 µl Mix, spin briefly PCR program PtMADS8 gene: 95 C 5 min 35 cycles: 95 C 30 sec; 55 C 30 sec; 72 C 1 min pro kb (calculate with the expected sizes for the PCR products) 72 C 10 min For pre-mirna amplification: Protocol will be given

10 Day 4 5. Herstellung chemisch kompetenter E. coli Zellen Zentraler Punkt jedes Klonierungsexperiments ist die Einführung von rekombinanter Plasmid-DNA in Bakterienzellen durch den Vorgang der Transformation. E. coli besitzt keine natürliche Transformationskompetenz und muss durch chemische oder physikalische Methoden künstlich kompetent gemacht werden. 1) Animpfen von 3 ml LB-Medium mit einem Klon von E. coli DH5α 2) Kultur über Nacht bei 37 C und 190 rpm schütteln 3) 50 ml LB-Medium werden mit 1 ml der Übernachtkultur angeimpft (in 200-ml-Kolben) Sie bekommen die vorbereitete Kultur und beginnen mit der Messung der OD600. 4) Schütteln ca. 2-3 h bei 37 C bis ein OD 600 von 0,5 bis 0,8 erreicht ist (jede halbe Stunde OD 600 messen!!!) 5) Ein Falcon (50 ml) auf Eis vorkühlen. 6) Kultur für 10 min bei 1118 g (5000 rpm) bei 4 C zentrifugieren 7) Ab jetzt alle Schritte auf Eis durchführen. 8) Überstand in den Kolben zurückgießen 9) Pellet in 5 ml eiskaltem 50 mm CaCl 2 resuspendieren 10) 10-minütiges Zentrifugieren der Kultur bei 1118 g (5000 rpm) bei 4 C 11) resuspendieren des Pellets in 20 ml eiskaltem 50 mm CaCl 2. Falcon immer auf Eis stehen lassen!! 12) 30 min auf Eis inkubieren 13) Kultur für 10 min bei 1118 g (5000 rpm) bei 4 C zentrifugieren 14) resuspendieren des Pellets in 2 ml eiskaltem 50 mm CaCl 2. Falcon immer auf Eis stehen lassen!! 15) 15 min auf Eis inkubieren 16) 300 µl eiskaltes 86 % Glycerin dazugeben 17) aliquotieren der Zellen (100 µl/eppi) und sofortiges Einfrieren in flüssigem Stickstoff 18) bei 80 C bis zur Transformation lagern Frage A) Wodurch erlangen die Zellen die Fähigkeit zur Aufnahme von DNA, bzw. wie wirkt CaCl 2?

11 Day 5 6. Aufreinigung der PCR Produkte mittels Gelextraktion Nach der PCR wird zur Analyse das Reaktionsprodukt auf einem Gel analysiert. Zum Entfernen von Nebenprodukten der PCR, und zum Entfernen der Primer, Polymerase und zum Pufferwechsel führt man eine Reinigung mittels Gelextraktion durch. 1) add 1x gel loading dye to the PCRs (stock solution: 6x) 2) load samples onto the gel, also run 5 µl of a DNA ladder mix 3) document the results 4) Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Cut the smallest possible gel slice!!! Place the gel slice into a 2 ml tube and weigh the gel slice using an empty tube as reference. 5) Add 300 µl binding buffer for every 100 mg agarose gel. 6) Incubate at 56 C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2 3 min during the incubation 7) Add 150 µl isopropanol for every 100 mg agarose gel to the sample and mix by vortexing. 8) Place one High Pure Filter Tube into one collection tube. 9) To bind DNA, pipette the sample (max. 700 µl) into the upper reservoir of the Filter Tube. If the volume of gel suspension is over 700 µl, simply load and spin again. 10) Centrifuge for 30 sec at full speed. 11) Remove the Filter Tube from the collection tube. Discard flow-through and place the Filter tube back in the same collection tube. 12) To wash, add 500 µl Wash Buffer and centrifuge for 30 sec. 13) Discard the flow-through and add 200 µl Wash Buffer, centrifuge for 30 sec. 14) Place the filter tube into a clean 1.5 ml microcentrifuge tube. 15) To elute DNA, add 50 μl ddh 2 O to the center of the Filter tube membrane, let the column stand for 1 min and centrifuge the column for 1 min at max.speed. 16) DNA Konzentrationsbestimmung durchführen (siehe oben) Frage A) Wie funktioniert die Reinigung der DNA mittels Silicamenbransäulchen?

12 Day 6 7. Restriction digests of the vectors and PCR products Zur Klonierung erfolgt nun ein Restriktionsverdau von Vektor und Plasmid. Zusätzlich wird der Vektor an den von den Restriktionsenzymen geöffneten Enden noch dephosphoryliert. Restriction digest of the vectors with simultaneous dephosphorylation pgreen + 35S 1 µg 10x Fast Digest buffer 1x FastDigest EcoRI 1 µl FastDigest HindIII 1 µl Fast AP (Alkaline Phosphatase) 1 µl ddh 2 O up to 20 µl Incubate for 15 min at 37 C pgreen + 35SmGFP5ER 1 µg 10x Fast Digest buffer 1x FastDigest SmaI 1 µl FastDigest BamHI 1 µl Fast AP (Alkaline Phosphatase) 1 µl ddh 2 O up to 20 µl Incubate for 15 min at 37 C

13 Digestion of PCR products (do 2 digestions for each PCR product) PCR product pre-mirna 10x Fast Digest buffer 200 ng 1x FastDigest EcoRI 1 µl FastDigest HindIII 1 µl ddh 2 O up to 30 µl Incubate for 15 min at 37 C PCR product PtMADS8 gene 200 ng 10x Fast Digest buffer 1x FastDigest SmaI 1 µl FastDigest BamHI 1 µl ddh 2 O up to 30 µl Fragen Incubate for 15 min at 37 C Combine the 2 digestions for each PCR product A) Warum wird der Vektor dephosphoryliert? B) Warum erfolgt die Klonierung mit zwei verschiedenen Restriktionsenzymen? Vom verdauten bzw. unverdauten Plasmid werden zur Kontrolle je 250 ng auf ein Gel 1% aufgetragen (Pro Gruppe 4 Proben). DNA 250 ng ddh 2 O auf 8 µl auffüllen 2 µl loading dye - 5 µl Marker neben die Proben auftragen - Gel bei 100 V ca 1 h laufen lassen 8. Purification of digested vectors and PCR products Zum Stopp des Restriktionsverdaus, zum Entfernen der Enzyme und zum Pufferwechsel führt man eine Reinigung der Reaktionsansätze mittels DNA-bindender Silicasäulchen durch. Beschriften sie ihre Röhrchen gut! 1) Adjust the volume of the DNA sample to be purified to 100 µl with ddh 2 O 2) Add 500 µl Binding Buffer to the DNA sample and mix well. 3) Place one High Pure Filter Tube into one collection tube. 4) To bind DNA, pipette the sample into the upper reservoir of the Filter Tube.

14 5) Centrifuge for 30 sec at full speed. 6) Remove the Filter Tube from the collection tube. Discard flow-through and place the Filter tube back in the same collection tube. 7) To wash, add 500 µl Wash Buffer and centrifuge for 30 sec. 8) Discard the flow-through and add 200 µl Wash Buffer, centrifuge for 30 sec. 9) Place the filter tube into a clean 1.5 ml microcentrifuge tube. 10) To elute DNA, add 50 μl ddh 2 O to the center of the Filter tube membrane, let the column stand for 1 min and centrifuge the column for 30 sec. 11) DNA Konzentrationsbestimmung siehe oben Frage A) Warum müssen die Restriktionsenzyme vor der Ligation aus dem Ansatz entfernt werden? Day 7 9. Ligation of the vectors with the PCR products In der Ligationsreaktion katalysiert die T4 DNA Ligase die Bildung von Phosphodiesterbindungen zwischen 5'-Phosphatenden und 3'-Hydroxylenden von DNA-Molekülen. Dabei hängt die Dauer und Effizienz der Reaktion nicht nur vom Enzym, sondern auch vom Vektor und den verwendeten Schnittstellen ab. Use the following equation for the calculation of the amount of insert used in the ligation reaction. ng vector (= 50 ng) kb size insert 3 = ng insert kb size vector 1 Important!!!The pre-mirna PCR product needs to be ligated to the vector pgreen + 35S. The target gene PtMADS8 PCR product needs to be ligated to the vector pgreen + 35SmGFP5ER. ligation Vector control vector 50 ng 50 ng insert DNA 3 : 1 molar ratio over vector - 10x T4 DNA Ligase Buffer 2 µl 2 µl PEG µl 2 µl T4 DNA Ligase 1 µl 1 µl ddh 2 O up to 20 µl FRAGE Incubate for 1 h at room temperature A) Warum verwendet man ein Verhältnis von 1:3 (Vektor:Insert) für die Ligation? B) Warum führt man eine Vektor Kontrolle bei der Ligation mit?

15 10. Preparation of the LB plates (cleanbench) - LB-Agar in der Mikrowelle schmelzen (Deckel der Flasche leicht öffnen, ständige Überwachung, da Gefahr von Siedverzug und Überkochen, Hitzschutzhandschuhe verwenden) - LB-Agar abkühlen lassen auf ca 50 C - Unter der cleanbench: Zugabe von Kanamycin (finale Konzentration: 50 µg/ml) - Flasche schwenken zum Vermischen - Platten zügig gießen (ca 25 ml pro Platte) - Platten halb geöffnet unter der Cleanbech trocknen lassen und beschriften 11. Transformation of the ligations into competent E. coli cells Mit dem erhaltenen Ligationsprodukt werden nun die zu Beginn des Praktikums hergestellten chemisch kompetenten Zellen transformiert. Dabei erfolgt auch eine Kontrolle der Zellen auf eine eventuelle Kontamination. Dabei wird statt der Ligation 10 µl Wasser zu den kompetenten Zellen gegeben und nach der Transformation alles auf eine Kanamycinhaltige LB Platte ausplattiert. thaw competent cells on ice add 10 µl of the ligation reaction to the competent cells and mix by flicking the tube incubate for 30 min on ice heat shock the cells for 1 min at 42 C, 2 min on ice add 1 ml of SOC medium incubate for 30 min at 37 C under shaking for the vector control and water control, centrifuge the cells for 1 min and remove as much supernatant, that approximately µl medium is left, resupend the cells, plate them out (cleanbench) for the ligations, first plate 200 µl, then centrifuge the cells for 1 min and remove as much supernatant, that approximately µl medium is left, resupend the cells, plate them out (cleanbench) incubate over night at 37 C

16 Day Analysis of plates from Transformation a. Count all the colonies on all your plates b. Evaluate and discuss your results! c. Choose 2 colonies from each construct for DNA-preparation (step 14) 13. Preparation of liquid E. coli cultures of the transformed cells (cleanbench) set up two liquid cultures of each transformed construct as follows: add 3 ml LB medium to a test tube, add Kanamycin to a concentration of 50 µg/ml (stock solution: 50 mg/ml) pick one colony with a yellow tip and transfer to the test tube incubate liquid cultures over night at 37 C 200 rpm cultures will be pelleted by centrifugation for 1 min and stored at -20 C until next practical course day This day, there will be a 2 h Bioinformatics part from Lydia Gramzow (additional script will be provided). Day Small-scale preparation of plasmid DNA from transformed E. coli 1) Place Binding Buffer on ice 2) Add 250 µl Suspension Buffer to the centrifuge tube containing the bacterial pellet 3) Resuspend the bacterial pellet and mix well 4) Add 250 µl Lysis Buffer 5) Mix gently by inverting the tube 5 times!!! To avoid shearing genomic DNA, do not vortex!! 6) Incubate for 5 min at Room temperature! 7) Add 350 µl chilled Binding Buffer 8) Mix gently by inverting the tube 5 times 9) Incubate on ice for 5 min (The solution should become cloudy and a precipitate should form) 10) Centrifuge for 10 min at approx. 13,000 x g (full speed) in a standard tabletop microcentrifuge 11) Insert one High Pure Filter Tube into one Collection Tube 12) Transfer entire supernatant from Step 10 into upper buffer reservoir of the Filter Tube 13) Insert the entire High Pure Tube assembly into the microcentrifuge 14) Centrifuge for 60 s at full speed 15) Remove the Filter Tube from the Collection Tube, discard the flowthrough liquid, and reinsert the Filter Tube in the same Collection Tube 16) Add 700 µl Wash Buffer II to the upper reservoir of the Filter Tube

17 Fragen 17) Centrifuge for 60 s at full speed and discard the flowthrough 18) Centrifuge the entire High Pure Tube assembly for an additional 30 to 60 s to remove residual Wash Buffer 19) Discard the Collection Tube 20) Insert the Filter Tube into a clean, sterile 1.5 ml microcentrifuge tube 21) Add 100 µl H 2 O to the upper reservoir of the Filter Tube 22) Centrifuge the tube assembly for 60 s at full speed 23) Determination of DNA-concentration A) Wie erfolgt die Lyse der Bakterienzellen? B) Worauf beruht das Prinzip der DNA-Bindung an die Silicamembran? C) Warum wird nur Plasmid-DNA, nicht aber genomische DNA mit dieser Methode gereinigt? 15. Restriction digest to check for the insertion of the PCR products Plasmid DNA 300 ng 10x Fast Digest buffer 1x FastDigest EcoRV 1 µl FastDigest BamHI 1 µl ddh 2 O up to 20 µl Incubate for 10 min at 37 C prepare also a sample of each construct without treatment with restriction enzymes add 1x gel loading dye to the samples (stock solution: 6x) load samples onto the gel, also run 5 µl of a DNA ladder mix document and evaluate the results QUESTION A) Which bands do you expect for digested and undigested plasmid? 16. Sequenzierung der Plasmide pgreen+ptmads8-gfp und pgreen+premirna Es wird eine Sanger-Sequenzierung der Plamide vorgenommen. Zur Bestimmung der Sequenz des Plasmids pgreen+ptmads8-gfp (Exon 4 bis Stop) und pgreen+premirna werden je 2 Ansätze pipettiert (fwd und rv Primer) pgreen+ptmads8-gfp pgreen+pre-mirna (Exon 4 bis Stop) DNA 120 ng DNA 120 ng

18 pgreen35s fwd pgreen35s rev pgreen35s fwd pgreen35s rev (5 µm) 0,5 µl (5 µm) 0,5 µl (5 µm) 0,5 µl (5 µm) 0,5 µl Mit Merck H 2 O auffüllen auf 8 µl Mit Merck H 2 O auffüllen auf 8 µl Für die Sequenzierung des Plasmids pgreen+ptmads8-gfp (Exon 2 bis Stop) werden 4 Reaktionen benötigt pgreen+ptmads8-gfp (Exon 2 bis Stop) DNA 120 ng pgreen35s fwd pgreen35s rev PtMADS8_fwd_XmaI_Exon4 PtMADS8_rev (5 µm) 0,5 µl (5 µm) 0,5 µl (5 µm) 0,5 µl (5 µm) 0,5 µl Mit Merck H 2 O auffüllen auf 8 µl Mit Merck H 2 O auffüllen auf 8 µl 17. Preparation of competent Agrobacteria GV3101 (will be provided) 1. Plate glycerol stock on YEB plates 2. Incubate for 2 days at 28 C 3. Inoculate a preculture (5 ml YEB media) with a single colonie 4. Incubate for h at 28 C 5. Inoculate a 500 ml culture (YEB media) with 1 ml of the preculture 6. Incubate for h at 28 C 7. Measure OD Let cells grow until OD600 of Precool centrifuge and centrifugation tubes 10. Centrifuge cells at 4 C, 10 min, x g 11. Wash pellet with 100 ml ice cold water 12. Centrifuge cells at 4 C, 10 min, x g 13. Repeat washing 4 times 14. Wash pellet with 10 ml ice cold water 15. Centrifuge cells at 4 C, 10 min, x g 16. Respend pellet with 1,5 ml ice cold sterile Glycerin 10 % 17. Aliquot cells à 40 µl 18. Shock freeze and store at -80 C Day Preparation of YEB plates 1. YEB-Agar in der Mikrowelle schmelzen (Deckel der Flasche leicht öffnen, ständige Überwachung, da Gefahr von Siedverzug und Überkochen, Hitzschutzhandschuhe verwenden) 2. YEB-Agar abkühlen lassen auf ca 50 C

19 3. Unter der cleanbench: Zugabe der Antibiotika Stock finale Konzentration µl Kanamycin 50 mg/ml 50 µg/ml Rifamycin 10 mg/ml 50 µg/ml Tetracyclin 8 mg/ml 2 µg/ml Streptomycin 100 mg/ml 50 µg/ml 4. Flasche schwenken zum Vermischen 5. Platten zügig gießen (ca 25 ml pro Platte) 6. Platten halb geöffnet unter der Cleanbech trocknen lassen und beschriften 19. Transformation of competent Agrobacteria using electroporation Um abzusichern, dass die Transformation, die Bildung des GFP und die gezielte Runterregulierung durch eine spezifische mirna in N.bentamiana funktionieren, werden verschiedene Kontrollen in den nächsten Experimenten benötigt. 1. pgreen + 35SmGFP5ER - Leerer Vektor nur mit Promotor und GFP = Zur Kontrolle der Expression des GFP 2. pgreen + 35S-PtMADS8-mGFP5ER - Eigener Vektor mit Promotor, Zielgen und GFP = Zur Kontrolle der Expression des Fusionsproteins 3. pgreen + 35S-AGL16-mGFP5ER - Vektor mit Promotor, Zielgen AGl16 und GFP = Zur Kontrolle der Expression des Fusionsproteins mit einem bekannten Zielgen der mir pgreen + 35S-AGL16-mGFP5ER in combination with its corresponding mirna pgreen + 35S-miR824 genomic - Positiv Kontrolle zur Interaktion von AGL16 und mirna pgreen + 35S-PtMADS8-mGFP5ER in combination with its putative mirna pgreen + 35S-Ptpre-miRNA - Nachweis der Interaktion der von ihnen klonierten mirna und target Gen 6. pgreen + 35S-PtMADS8-mGFP5ER in combination with a mirna not targeting GmMADSX pgreen + 35S-miR160a - Negative Kontrolle, es findet keine Interaktion statt 7. pgreen + 35S-PtMADS8-mGFP5ER in combination with the empty pgreen + 35S vector - Negative Kontrolle, es findet keine Interaktion statt These constructs are to be transformed: o pgreen + 35SmGFP5ER o pgreen + 35S- PtMADS8-mGFP5ER

20 o pgreen + 35S-Ptpre-miRNA o pgreen + 35S-AGL16-mGFP5ER o pgreen + 35S-miR824 genomic o pgreen + 35S-miR160a o pgreen + 35S Gruppe Gruppe Gruppe Gruppe 7 Gruppe 8 Gruppe 9 Gruppe 10 o pgreen + 35SmGFP5ER o pgreen + 35S-PtMADS8- mgfp5er o pgreen + 35S-Ptpre-miRNA o pgreen + 35S-AGL16- mgfp5er o pgreen + 35S-miR824 genomic o pgreen + 35S-miR160a o pgreen + 35S thaw competent cells on ice add 400 ng plasmid DNA and mix transfer cells into a pre-chilled electroporation kuvette incubate for 5 min on ice electroporation at 1,8 kv add quickly 1 ml YEB medium and transfer cells into a 1.5 ml tube incubate for 1.5 h at 28 C under shaking centrifuge for 1 min and remove as much supernatant that around µl is left resuspend cells plate all onto YEB plates incubate for 2 days at 28 C QUESTIONS A) What is Agrobacteria tumefaciens? Why is it used for transformation of plant material?

21 Day Transformation of Nicotiana benthamiana leaves Each group will be informed which experiments they will perform. Use different Agrobacteria combinations: 1. pgreen + 35SmGFP5ER OD 600 = 0.5 (as positive control) 2. pgreen + 35S-PtMADS8-mGFP5ER OD 600 = 0.5 (as positive control) 3. pgreen + 35S-AGL16-mGFP5ER OD 600 = 0.5 (as positive control) 4. pgreen + 35S-AGL16-mGFP5ER in combination with its corresponding mirna pgreen + 35S-miR824 genomic in different OD combinations (positive control experiment for the interaction of AGL16 and mirna824): pgreen + 35S-AGL16-mGFP5ER pgreen + 35S-miR824 genomic OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.1 OD 600 = 0.5 OD 600 = 0.05 OD 600 = 0.5 OD 600 = pgreen + 35S-PtMADS8-mGFP5ER in combination with its putative mirna pgreen + 35S-Ptpre-miRNA in different OD combinations: pgreen + 35S-PtMADS8-mGFP5ER pgreen + 35S-Pt-miRNA OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.1 OD 600 = 0.5 OD 600 = 0.05 OD 600 = 0.5 OD 600 = pgreen + 35S-PtMADS8-mGFP5ER in combination with a mirna not targeting PtMADS8 pgreen + 35S-miR160a in different OD combinations (negative control): pgreen + 35S-PtMADS8-mGFP5ER pgreen + 35S-miR160a OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.5 OD 600 = pgreen + 35S-PtMADS8-mGFP5ER in combination with the empty pgreen + 35S vector in different OD combinations (negative control): pgreen + 35S-PtMADS8-mGFP5ER pgreen + 35S OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.5 OD 600 = 0.1 inoculate 50 ml YEB medium containing 50 µg/ml Rifampicin, 2 µg/ml Tetracyclin and 50 µg/ml Kanamycin (stock solutions: Rifampicin 10 mg/ml, Tetracyclin 8 mg/ml, Kanamycin 50 mg/ml) with one colony of transformed Agrobacteria incubate over night at 28 C at 200 rpm (will be provided) prepare 200 ml of infiltration medium (10 mm MgCl 2, 150 µm acetosyringone), stock solutions are 1 M MgCl 2 and 150 mm acetosyringone

22 measure OD 600 of the cells (they should be around OD 600 = 1.0) transfer the cells into 50 ml falcon tubes (with the violet cap), and centrifuge them for 3 min at rpm discard the supernatant into an S1 waste flask wash the cells with 40 ml infiltration medium to remove traces of antibiotics centrifuge again for 3 min at rpm, discard the supernatant resupend the cells in infiltration medium so that an OD 600 = 0.5 is reached measure OD 600 again to check incubate the cells in infiltration medium for 1.5 h at 28 C with 200 rpm shaking combine the different Agrobacteria solutions as described above, end volume is 10 ml choose N. benthamiana plants (label them) and two leaves on each plant (they have to be fully expanded), mark the leaves with a tape, inoculate 2 plants/combination fill the Agrobacteria solution into a 3 ml syringe without a needle and infiltrate the underside of the leaf without damaging it (use protective googles) Day Visualisation of the GFP signal using a microscope with GFP3 filter Gruppe Datum + Zeit

23 Appendix target gene CDS >PtMADS8_genomic Atggggagggggaagatagtgataagaaggattgataactcgacaagcaggcaagtgaccttctcaaa gaggaggaatggattgttgaagaaagcgaaagaactggcgatcctttgcgacgcagaagttggagtca tgatcttctctagcaccggcaagctctatgatttctccagcaccagcatgaaatcagtcattgaaaga tacaacaaatcaaaagacgagcatcatcaaatgggcaacccaacctctgaagtcaagtttttaaagca ttgcatttttgtcaaagatagctccagatacattactaggaatcaccatgtggaactttgccttttct gctttaaacaatgctgttaccacgagcaggatggatttttttggcgattgcaaatgatgggtgaacaa ctttctggcttaagcgtaacagatttgcagaatttggagagccaattggaaatgagtcttcagggtgt tcgcatgaaaaaggaccaaattttaatggatcaaatacaagaactaaatagaaagggcaacctcattc accaagaaaatgttgaactctatcagaaggtctatggaacaagggacgtaaacagagcaaacagaaat tcccttctcacaaatggtctggccatcggagaggaatcacatgtacctgtccatctccagcttagcca gccacaacaacaaaaccatgatacaccagctacaaagttggggtacaatttcctccacagcacaagta tttaa Predicted protein sequence MGRGKIVIRRIDNSTSRQVTFSKRRNGLLKKAKELAILCDAEVGVMIFSSTGKLYDFSSTSMKSVIERYNKSKDE HHQMGNPTSEVKFLKHCIFVKDSSRYITRNHHVELCLFCFKQCCYHEQDGFFWRLQMMGEQLSGLSVTDLQNLES QLEMSLQGVRMKKDQILMDQIQELNRKGNLIHQENVELYQKVYGTRDVNRANRNSLLTNGLAIGEESHVPVHLQL SQPQQQNHDTPATKLGYNFLHSTSI- Exon Intron Structure 1kb Genomic sequence of PtMADS8 from 2 nd Exon Exons in grey, predicted mirna-target site in pink Exon 1... Intron 1... catgaaatcagtcattgaaagatacaacaaatcaaaagacgagcatcatcaaatgggcaacccaacct ctgaagtcaaggtatttcagaaaccgattttcattttattttgcgagtttgtgcaaaatttgattcgt cgtattattttccacatttgcatatagaatttagttatacatcaaaacgaacatgttaaattaaattt actacttgtagtggaacagcttacacatctctagtttttaaagcattgcatttttgtcaaagatagct ccagatacattactaggaatcaccatgtggaactttgccttttctgctttaaacaatgctgttaccac gagcaggatggatttttttggcgattgttacagctaaaagatactgcatctaacaattctgcatttca gctaaaagtttgcacctattggacgagctatacttgcaaacataacattatgacaccgaagggttgag atattttgttttataaaacatgttaataacgtaagaaaatcttaatgtttctgatcatttatcagcag gatgtgctaataaacatggacttatgaatggaaatctaactaaataaattatccggcataatgtggca ataaagaagggaaatgaagatacaatttgtacagactacagataagctatgccacatctaggacttgg Exon2 Exon3

24 cctttaatatgatttcagaaaggcataagagccccttgaacatgcaagtataaaattggtatggcaaa atgttgcaaatgacaaaaccagtataagtcctaattctaatactgctgaaggaaataacatatcaagt gtttagtccaaaatgatttaggaataagaaacagaaaccaattaatattccatgactggattctgata tttaaagcacatgataaataaaaaaaagttagacaacggcaatggttctgccggttattagctgcatg aaaatcctatctactatagatatcctcaaatttaaaagcagcagataatgactggcaagattgtgcaa cacaatcaacaaatgacttgcatttgaaacagcacaataacacatatcataggttaataagagagtat gcataatgaagttgtttctaccacagaaatgtacttcattaagcttcaaagtgtagacaggaaatcat ctgtgttattgttctaaattagcaggataaactgaaattaaaaacacaagtatatgtcaactaacatg tcatgtcttcaggctccagcaaagcacggcccaattgaataaataaatacgcatatgataaagcatac gaactccacacaaccaagccttattgattcattcaagctttaaggtgtagcatcatatcttacactgg acctctgctaaccgcaactgtcctcctttatctgcaatgggattgggaccatctgcgtgcagatagaa ctcacaagacattttatcagtagcaagaaaatacatgtcatcttgaagcacgtcatagtctattaaaa atgctttctagttttgtcgagataaaggtagagctacatggaaggttagagagggggtggtattcaga catgaaaaagaaaaaatatggtgtattgaatcataatcactatatggagattagacagtggcttatca gaatgaaaaggatgattttttttatccctttttcttcagatgtgttttcataacagcagaaggcaatt actcttcattttagtgaatgcatgttaaccacaaaagttggtaattccactaaattgtgcattatatt ttgctagttctggcaaagggaggcagcagtgttgagacagcaactgcaaaccttgcaagaaaatcatc ggtatgatccattcccttcaaacaatagtgttttatatgaagataaatgaacataaagactgaaaata gaataattgcgacagatcaaatataaaaaactgaaactttcaaaggagcatgctattatttaagtcag acacttgcaggtggtactggtgtccatgtgatcactaacaatttagacacaaacacaaaaaaaatcac ttgtgtcacactgaaaaagttcatctcggtcacctctaagaaatgtcagcattgtgcatcaggagcaa aaggggccacaagaaggcagcaaccatacaccctctcctaaaagtattcaagtagtactagtcaggtt tatgaccccaagaaatttatcgcagagttaacccgaagactttcaagccaagtcacatttacaagcat ttttgaaattcttgttatcgaacaagattaaagcatctatatgaacacatacctataaatacctctag tatcacgtctacagctgaacaagtcatttctccaattatcttttgcccatctatttcttcctagtaat atcttcttttttcatcaggaaaagaatgaaaagcattctagagtcatttctccaatgtgaataaacta cctctagccattgataccccaaactaagggtcactccaaaatatatatataactcaaaacaaagacta acagtaaatcttaccggaacataaaaggaagcatccaagatagctaatgctgttggaaaaatccttca ggaacagaaaacagtaaaaagattaaatggatcattgcttctcagtacatgcgtcattagcaaagttt acaatgtcctagacaaaagtttaaaacctaactgcatttccatatgatgataacaggcaaatgatggg tgaacaactttctggcttaagcgtaacagatttgcagaatttggagagccaattggaaatgagtcttc agggtgttcgcatgaaaaaggtttgaaattcctacaaccacagtgcagtctttagatttccctaaagt ggaacacatccatctcactgaactgatattgacttcatcaggaccaaattttaatggatcaaatacaa gaactaaatagaaaggtaaacctgtaaacaaaggctaccgacttgctttgtaaagtttttttaattca aatagctaaactttgatatatctgttgcagggcaacctcattcaccaagaaaatgttgaactctatca gaaggtaaactatttcgtcatgaaaatcaagaattgtataaattataagtttgagtaagcagaaactt ataagatccttgcaacttattgattcaaaccatcactaccttagtcctgaatcaaattgcaaggcgtt tcattccaccctcattagcatattttcacatcccaggtctatggaacaagggacgtaaacagagcaaa cagaaattcccttctcacaaatggtctggccatcggagaggaatcacatgtacctgtccatctccagc ttagccagccacaacaacaaaaccatgatacaccagctacaaagttggggta caatttcctccacagcacaagtatt taa Primers PtMADS8_fwd_XmaI_Exon2 CATCAT CCCGGG atgaaatcagtcattgaaagatacaac Exon7 Exon6 Exon5 Exon4

25 PtMADS8_fwd_XmaI_Exon4 CATCAT CCCGGG atgatgggtgaacaactttctg PtMADS8_rev_BamHI GA TACG GG ATC C aatacttgtgctgtggaggaaattg premirna Tttatagtcaaaaagctatagaagccctgttgaaatttggtccctttatagacccaagaa (Intron ~22 kb) Ggaccaaatttcaacagggattctacagctttttgactataaa Primers Pt_fwd_EcoRI_miRNA GCCAGT GAATTC Tttatagtcaaaaagctatagaagcc Pt_rev_miRNA_part1 cctgttgaaatttggtcc ttcttgggtctataaagggacc Pt_rev_HindIII_miRNA AGCTCG TTCGAA tttatagtcaaaaagctgtagaatccctgttgaaatttggtcc Pt_rev_HindIII_miRNA_short AGCTCG TTCGAA tttatagtcaaaaagc

26 pgreen + 35S (4721 bp) for cloning putative pre-mirna

27 pgreen + 35SmGFP5ER (5519 bp) for cloning mirna target gene in frame with GFP

28 YEB-Medium (ph 7,2): 5 g Rinderextrakt 5 g Trypton 5 g Saccharose 1 g Hefeextrakt 0,5 g MgSO 4 x 7H 2 O ad 1 Liter dh 2 O DNA-ruler