How are AAC blocks pest resistant

Transformation and molecular genetic analysis of the diatom Cylindrotheca fusiformis

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1 Large-scale biochemical internship at the University of Regensburg, part C: Molecular biology, transformation and molecular genetic analysis of the diatom Cylinderotheca fusiformis as of December 07

2 Contents 1. Introduction General Practical Work Cloning of a Resistance Gene for Diatoms Transformation Plasmid Isolation [Machery Nagel; NucleoSpin Plasmid] AccI / KpnI digestion of ppδ or pblen Ligation: ppδ (AccI / KpnI) with blen (AccI / KpnI) transformation in E. coli colony PCR (identification of clones which contain the ble gene) Prepare the Plasmids ppδblen for the diatoms transformation Transformation of C. fusiformis Molecular genetic analysis of a C. fusiformis transformant RT-PCR analysis of CfWT and the transformant Cfε and cloning of the cdna amplicon Sequencing of the PCR product Southern blot with restriction-digested genomic DNA from Cf WT and the Transformant Cfε Quantitative PCR with genomic DNA from CfWT and Cfε Appendix Composition of the solutions used DNA marker DNA sequences Literature: W (WW) worthwhile ... 28

3 1. Introduction 1.1 The general aim of this part of the internship is to provide an insight into molecular biology work and routine techniques. The spectrum includes cloning in E. coli; preparative isolation of a plasmid and subsequent transformation of this transgenic DNA into diatoms. In the second part, a molecular biological analysis of a diatom mutant is carried out. In addition to the internship, there are seminars that are intended to explain the respective subject areas in more detail. These seminars are held by both assistants and internship participants. The seminar topics are flexible and can be tailored to the general interest. The following examples serve as a guide: Molecular biology enzymes and the reactions they catalyze Molecular biological model organisms PCR: Applications / methods Hybridization technologies: Applications / methods Nucleic acids: Types / characteristics / functions ... At the end of the internship, the results are summarized in the form of a protocol . The protocol is structured as follows: Introduction Brief description / classification of the experimental systems used and consideration of the "project" in the "larger framework" of the molecular biological analysis of diatoms. Experimental part The experimental goal of each sub-step is named. The procedure is described without going into the details given in the script (e.g. quantity / temperature information, centrifuge times) [no copying / pasting]. Reference is made to the standard protocols used and these are noted together with the reagents used in the annex. If the specifications in the scriptum are changed, this must be recorded (please also state the reason why the protocol was changed). All (important) observations are noted and (partial) results are documented in the form of the original data. Summary Brief assessment of the work carried out and discussion of how (or if not, why not) the experimental goals were achieved. Annex Contains standard protocols, reagents, buffer compositions, etc. 1

4 1.2 Practical work Buffers and solutions from in-house production At the beginning of the internship, buffers and solutions are prepared. In the past it has been shown that this (unfortunately only moderately exciting) task was undertaken by the students in a joint effort. Unfortunately, these "joint projects" repeatedly led to the collective failure of entire parts of the internship, which had to be repeated afterwards. The correct buffer composition is essential for the success of the experiments. Each group has to prepare its own buffers. Stock solutions issued As part of the internship, the assistants will issue special stock solutions and reagents. These are often concentrates: These have to be diluted accordingly in order to obtain the correct concentrations in the reaction batches. If the labeling of the dispensed materials is ambiguous, please ask the assistants before pipetting. Sterile work All sterile work (including plating of transformed bacteria) is carried out on the sterile bench. If preliminary stages (e.g. ligation approaches) are placed on the laboratory bench, it should be wiped off beforehand and cleaned with 70% ethanol. Pipetting work In many parts of the practical course, very sensitive methods are used (e.g. PCR / transformation) in which contaminations that have been introduced have a negative impact on the results obtained. New pipette tips must always be used when pipetting from stock solutions. If the pipette tip touches the bench or other objects (unintentionally) before the pipetting process, these should be discarded. Labeling At the beginning of the internship, each group should come up with a clear labeling strategy for the samples that arise during the course of the internship. This avoids mix-ups and losses. 2

5 2 Cloning of a Resistance Gene for Diatom Transformation Two plasmids (ppδ and pblen) are available for the synthesis of the resistance gene. The plasmid ppδ contains a diatom-specific promoter Pδ (Pdelta; approx. 950 bp, FIG. 1A). Plasmid pblen carries the bacterial ble gene (BLE; approx. 380 bp), which mediates resistance to the antibiotic Zeocin (Fig. 1B). N denotes a diatom-specific 3 'non-transcribed region (N; approx. 575 bp) which immediately follows the stop codon of the ble-gene and which contains the terminator of the C. fusiformis fruα2 gene. After restriction digestion, the DNA fragment blen is isolated from pblen and ligated with the ppδ vector digested by appropriate restriction enzymes (FIG. 1C). The ligation product (ppδblen) is cloned in E. coli, then isolated and used for the transformation of the C. fusiformis diatom. C. fusiformis clones which have been successfully transformed and which express the ble-gene can grow on medium containing Zeocin. A Pdelta B Acc I (1067) ampr ppdelta 5312 bp ampr pblen 4865 bp Acc I (1067) BLE N KpnI (2023) C Kpn I (2470) BamHI (104) ClaI (124) pdelta X031 ampr ppdelta blen 4865 bp Acc I (1067) BLE BLE6 ClaI (1664) N KpnI (2023) Eco RI (2031) ClaI (2038) Fig. 1 Plasmid maps of the original vectors (A, B) and the target plasmid (C) with the relevant elements (described in the text), Restriction enzyme sites and primer binding sites. ampr codes for the enzyme β-lactamase, which gives plasmid-bearing bacteria resistance to ampicillin. 3

6 2.1 Plasmid isolation [Machery Nagel; NucleoSpin plasmid] 1. for each of the two clones (ppδ [GK16] or pblen [GK17]) 10 ml above-mentioned culture (37 C / 190 rpm, in 2xYT / Amp medium) 2. The cells are for Pelleted for 10 min at 2800xg ml and 4 C 3. Remove the supernatant completely 4. See protocol in Appendix 5. Photometric determination of the DNA concentration and the ratio E 260 / E AccI / KpnI digestion of ppδ or pblen 1. 2 µg ppδ or 5 µg pblen are in 1x NEB4 buffer including 0.1 mg / ml BSA with 15 U KpnI (ppδ) / 30 U KpnI (pblen) and + 15 U AccI (ppδ) / 30 U AccI (pblen) in a total volume of 100 µl digested C / 2h 3. analyze 10 µl of each digest on a 1% agarose gel; if digestion is complete, then ethanol precipitation: 1. add 1/10 vol. 3M NaAc ph 5.2 + 2.5 vol. EtOH to the aqueous phase 2. mix as above min, 20 ° C. 4. centrifuge: 10 min / 14000 rpm / 4 C, discard the supernatant 5. Pipette 100 µl 70% EtOH into the pellet (if possible do not swirl the pellet) 6. Centrifuge for 5 min at 4 C, discard the supernatant 7. Dry the pellet at 37 C 8. Dissolve in 30 µl TE; separate by electrophoresis (1% agarose) (divide into three pockets for each digestion mixture) 9. Cut out the 3.9 kb band (ppδ) or 950 bp band (blen) (use only 366 nm UV lamp for detection!) and use Isolate QiaExII (10 µl glass milk) (elution in 10 µl TE) [see protocol in the appendix] Quantity estimation of 1/10 of the eluate using agarose gel electrophoresis 2.3 Ligation: ppδ (AccI / KpnI) with blen (AccI / KpnI) L1 L2 blen (AccI / KpnI) 25 ng ---- ppδ (AccI / KpnI) 100 ng 100 ng + 10x T4 DNA ligase buffer 2 µl 2 µl + 10 mm ATP 2 µl 2 µl + T4 DNA ligase 2 U 2 U + H2O to 20 µl to 20 µl at 12 C above sea level incubate (if the ends are sticky, a ligation at RT can alternatively be carried out for 1-2 hours on the laboratory bench) 2.4 Transformation in E. coli Production of competent cells: 1. Preculture of E. coli DH5α [GK7] in 2 ml LB medium overnight 170 rpm / 37 C 2. inoculate the main culture with it (2x 50 ml LB in 250 ml flasks) 3. shake at at least 170 rpm / 37 C until OD 578 = 0.5 (growth time approx. 2 h) 4. in sterile 50 ml Pour Falcon tubes and centrifuge: 7 min / 2000 g / 4 C 5. Discard the supernatant 6. Resuspend cells in 15 ml TfbI (vortex vigorously!) Min / 0 C 8. Centrifuge: 5 min / 1500 g / 4 C 9. Discard the supernatant 10. Resuspend cells in 2 ml TfbII (careful swirling in ice water!), Fill µl aliquots into sterile Eppendorf tubes (pre-cooled with liquid N2) (let cells stand on ice!), Freeze immediately in liquid N2 and at -80 ° C to store. 4th

7 2.4.2 Transformation: Fill µl of competent E. coli DH5α (thaw in your hand, then immediately on ice) into ice-cold E-cups [50 µl of competent E. coli (given by the assistant) as a control transform only with 1 ng of ppδ ( see 2)] µl ligation mixture L1 or L2 (negative control) or 1 ng ppδ (positive control) min / 0 C sec / 42 C 5. 2 min / 0 C µl SOB medium, 5 µl 2M MgCl 2 ( sterile!) µl 1M glucose (sterile!) min / 37 C 10. Pellet 2 plates each (100 µl or remainder by centrifugation [5 min .; rpm (xg)] for L1 or L2 batches and absorb in 100 µl supernatant ) plate out on LB / Amp plates; of the positive control only 11. plate out 1x 100 µl 12. u. N. / 37 C the rest of the ligation is analyzed in a 1% agarose gel together with the gel-purified ppδ blen fragments the positive controls are used to determine the cfu (the colony forming units) per µg of transformed DNA [measure of the competence of the cells] . 2.5 Colony PCR (identification of clones that contain the ble-gene) x-PCR mix (for eleven 50 µl reactions): 27.5 µl l 2mM dntp-mix (each contains 2mM datp, dctp, dgtp, dttp) 110 µl 5x PCR buffer (provided) 27.5 µl 10 pmol / µl X031 primer 27.5 µl 10 pmol / µl BLE6 primer 22 µl DMSO X µl Taq Pol. (Ask your supervisor) to store 275 µl of water in ice 1. For 10 clones: pick up approx. Half of a clone from the transformation plate with sterile pipette tips, resuspend in 25 µl water (in a PCR cup) and transfer the cells remaining on the pipette tip to LB / Apply amp plate. Plate above incubate. 2. Cups: 5 min 95 C, immediately to 0 C, briefly centrifuge at 4 C (adapter!) Μl 2x Mix PCR program Keep samples on ice until the thermoblock of the PCR machine has reached 94 C Place the sample vessels in the block Step 1 Denaturation 2 min, 94 C 2 denaturation 30 sec, 94 C 3 annealing 30 sec, 55 C 4 extension 45 sec, 72 C 5 cycles go 40x back to 2 6 extension 7 min, 72 C 7 storage 4 C 10 μl each of the batches analyze on 1.5% agarose gel (expected product: 584 bp) 5

8 2.6 Preparing the plasmid ppδblen for the diatom transformation Plasmid isolation [Machery Nagel; NucleoSpin Plasmid]: 1. 10 ml each over the top. Culture (LB / Amp) of 2 clones positive in the colony PCR. Isolation of the plasmids from each 5 ml culture using the NucleoSpin kit (see protocol in the appendix) 2. Photometric determination of the DNA concentration and the E 260 / E ratio If DNA concentration <1 µg / µl: ethanol precipitation: 1. as under 2. Dry the pellet at 37 C 3. Dissolve in x µl 10mM TRIS-Cl ph8 to obtain a 1 µg / µl solution 4. Take 2.5 µl plasmid solution 5. +2.5 µl TE µl 6x DNA -Sample buffer (DNA-PP) quantity estimation in 1% agarose gel (1x TAE) restriction analysis (see 2.2) In independent 20 µl batches (final volume) 0.5 µg plasmid DNA (for a and b) or 1 µg plasmid DNA are added (for c) digested as follows: 5 U KpnI (1x NEB1 + 0.1 mg / ml BSA) 4.9 kb band 5 U BamHI / 5 U EcoRI in EcoRI buffer with 0.1 mg / ml BSA 1.9 kb band + 3.0 kb band 10 U ClaI in NEB4 + BSA (100 mg / ml) 0.35 kb + 1.55 kb + 3.0 kb band Adsorption of the plasmid DNA on tungsten particles Washing of the tungsten particles: mg tungsten - Resuspend the particles in 500 µl ethanol pa 2. 1 min z centrifuge 3. pellet in 250 µl ethanol p. a. resuspend min pre-tex 5. centrifuge for 1 min 6. wash pellet 3 times with 250 µl sterile H 2 O each time (resuspend each time, centrifuge for 1 min), after the last centrifugation in 150 µl H 2 O, resuspend the suspension in 50 µl aliquots (sterile 1.5 ml cups) Coating: 1. Add to 50 µl aliquots with continuous pretexting: 5 µl 1 µg / µl plasmid DNA 50 µl 2.5 M CaCl 2 (sterile) 20 µl 0.1 M spermidine ( free base, sterile) 2. then centrifuge for 3 min pre-text sec, remove supernatant completely 4. pellet in 250 µl ethanol p. a. resuspend 5. Centrifuge 1 min pre-tex sec, pellet in 50 µl ethanol p. a. resuspend keep suspension on ice until transformation 6

9 2.7 Transformation of C. fusiformis Schematic representation of the BioListic system used for transformation (PDS-1000 / He system) rupture disk rupture disk macro carrier macro carrier stopping screen stopping screen Preparing the cells Harvesting C. fusiformis wild-type cells: 1. pro Apply the group to 2 ASW plates per cells, to do this: 2. Determine the cell density of the culture (counting chamber) Centrifuge cells in sterile 50 ml Falcon for 10 min at 2800 g, RT 4. Resuspend the pellet in a little ASW medium 5. Half of each of the cell suspension in the central region (Ø approx. 5 cm) of an ASW plate 6. Allow to dry for approx. 30 min (sterile bench) Bombardment of the cells Pretreatment: 1. "Rupture disc" and "Microcarrier plate" in ethanol p. A. Insert, shake off the ethanol and let it dry standing in a sterile Petri dish under a sterile bench; Anneal the stopping screen and microcarrier holder for 4 hours at 200 ° C 2. Coat the central area of ​​a "microcarrier plate" with 10 µl tungsten particles (DNA coated) as evenly as possible, allow it to dry for 10 minutes 3. Install the microcarrier plate in the holder ("stopping - Don't forget the screen "!) 4. Install the" rupture disk "in the" cannon "[Biolistic Particle Delivery System PDS-1000 / He-Particle-Gun (BioRad)] 5. Distance rupture disc / stopping screen = 1, 5 cm 6. Place the agar plate in the interior of the "cannon"; Distance rupture disc / agar plate = 7 cm 7. Evacuate the apparatus 8. Blast the plate with He as the pressurized gas at 1100 psi, 1350 psi or 1550 psi 9. Bring the apparatus to atmospheric pressure and remove the agar plate. Incubate under constant light in the culture room (18 C) for h selection Zeocin-resistant C. fusiformis cells 1. Rinse two plates (one group) each time with ASW medium, so that at the end approx. 1 ml cell suspension is obtained. 7th

Spread 10 2. 1/5 of the cell suspension, approx. 200 µl on 1 plate of ASW + Zeocin (1 mg / ml) (corresponds to approx. 10 7 cells). 3. Incubate in the light for approx. 7 days / 20 C 4. "Pick" 6 independent clones individually using a toothpick and inoculate 1.5 ml ASW + 1 mg / ml Zeocin medium on the titer plate; In parallel to this, inoculate 1 well with the transformant Cfε and inoculate 1 well (ASW without Zeocin!) with C. fusiformis wild type (CfWT) for approx. 6 days / 20 C incubate in the light. PCR analysis of Zeocin-resistant cells 1. As soon as titer plate cultures have grown are (after approx. 6 days), remove 1 ml of cell suspension each 2. Centrifuge in 1.5 ml E-Cup (3 min, table centrifuge) 3. Remove the supernatant up to 200 µl, rinse the inside wall of the cup thoroughly with the remaining supernatant 4. Centrifuge as above, remove the supernatant completely 5. Resuspend the pellet by pipetting up and down in 20 µl DNA lysis buffer (see Appendix) 6. 5 min 95 C, then immediately with 200 µl 100 mm Tris HCl pH 7.5 dilute 7. Centrifuge for 5 min, 1 µl of the supernatant + 49 µl PCR mix, briefly "pre-text" 8. Immediately place in thermal cycler / 55 C PCR mix (for nine 50 µl reactions): prepare shortly after cell lysis and keep standing on ice 22.5 µl 2mM dntp-mix 90 µl 5x PCR buffer 22.5 µl X031 primer (10 pmol / µl) 22.5 µl BLE6 primer (10 pmol / µl) 11.5 µl DMSO 4.5 µl Taq Pol 267.5 µl H2O PCR program: keep samples on ice until the thermoblock of the PCR machine has reached 94 C. Put the sample vessels in the block set step 1 denaturation 2 min, 94 C 2 denaturation 30 sec, 94 C 3 annealing 30 sec, 55 C 4 extension 45 sec, 72 C 5 cycles go 40x back to 2 6 extension 7 min, 72 C 7 storage 4 C gel electrophoresis ( 1.5% agarose) of 10 µl each (expected product: 584 bp) 8

11 3 Molecular genetic analysis of a C. fusiformis transformant In a previous transformation experiment, C. fusiformis was transformed with the fruε gene of the diatom Navicula pelliculosa (encoded for the cell wall protein ε-frustulin). Southern blot and quantitative PCR should be used to check whether and how many copies of the fruε gene have been integrated into the genome of C. fusiformis. In order to prove that the fruε gene is transcribed in the transformant and the pre-mrna (contains three introns) is correctly processed, RT-PCR analyzes are carried out and the resulting PCR products are cloned and sequenced.A intron 1 R16 NcoI (351) exon 2 fru epsilon X008 exon 1 R17 X013 intron 2 exon 3 intron 3 exon 4 EcoRI (1408) frustulin 1450 bp fru alpha 2 fru alpha 2 B fru alpha 2 fru alpha 2 fru alpha 2 fru alpha 2 R15 fru alpha 2 R14 fru alpha 2 CF FA2G 4808 bp CP (BLA) ALPHA D Xmn I (2299) uni Bam HI (430) Sma I (437) ampr puc bp rev Eco RI (451) P (LAC) ORI Fig 2A) Fruε gene from N.pelliculosa: the positions of introns, exons, primer binding sites and an Eco RI recognition sequence are indicated. qpcr primers are highlighted in red. B) Fruα2 gene from C. fusiformis: functional elements are noted as described under A). C) Plasmid map of the puc18 vector. Functional elements, primer binding sites (uni / rev) and relevant restriction enzyme sites are indicated. P (BLA), P (LAC) are prokaryotic promoters. ALPHA denotes the lacz ORF. ORI is the origin of replication. ampr, see Fig. 1. D) Example gel for the electrophoretic separation of RNA from C. fusiformis. Left lane, λ DNA-Eco130I standard. Middle lanes, 2 independent RNA preparations. Right lane, 100bp DNA standard. 9

12 3.1 RT-PCR analysis of CfWT and the transformant Cfε and cloning of the cdna amplicon 1. RNA is unstable and RNAses everywhere 2. Put / pack and autoclave pipette tips and Eppendorf tubes especially for this part of the lab 3. DEPC-treated solutions use RNA isolation 1 Inoculate 250 ml of ASW medium each with CfWT or Cfε and allow to grow to a cell density of about 10 6 cells / ml 2. Centrifuge 50 ml of culture (5 × 10 7 cells) in 50 ml Falcon tubes (10 min / 2800 g / RT) 3. Resuspend the cell pellet in 1 ml ASW medium and centrifuge it in a sterile 1.5 ml cup (2 min / g / RT), remove the supernatant completely. 4. Put the cell pellet in 1 ml TRI REAGENT (contains guanidine isothiocyanate and phenol) by repeated pipette up and down thoroughly resuspend 5. Let stand 5 min at RT Add µl chloroform and shake vigorously for 15 sec Let stand min at RT, then centrifuge (15 min / g / 4 C) 8. Aqueous phase (above; not int erphase or organ. Phase with it!) Transfer into a new 1.5 ml cup, add 500 µl isopropanol and pre-text; 9. Leave to stand for 5 min at RT, then centrifuge (10 min / g / 4 C) 10. Carefully remove the supernatant, wash the pellet with 1 ml 70% EtOH (pre-text) 11. Centrifuge (10 min / g / 4 C) ), Remove the supernatant, centrifuge again for 1 min, remove the supernatant completely 12. Dry the cup with the lid open at RT for 5-10 min (until no more EtOH odor can be felt; do not dry too long !!!) 13. Dried pellet in 20 µl each Take up H2O (DEPC-treated), 10 min 65 C, then immediately put on ice µl are analyzed immediately for quality control using gel electrophoresis [+ 5 µl H2O (DEPC-treated), + 1 µl 6x DNA sample buffer (fresh!); 1.5% agarose] 15. If the RNA is OK, then take the aliquot (ask assistant!) For RT-PCR 16. Remaining RNA solution: +1/4 vol. 10M LiCl (DEPC-treated), store at 4 ° C. Reverse Transcription with subsequent PCR (RT-PCR) each for CfWT and Cfε x µl RNA preparation (ask assistant!) + 1 µl RNase inhibitor + 4 µl 5x First-Strand-Buffer (will be provided) + 1 µl 10 mm dntps (will be provided) + 1 µl 100 mm DTT (provided) + 2 µl 10 pmol / µl ON670 ad 19.5 µl H2O (DEPC-treated) + 0.5 µl 200 U / µl RevertAid RTase Step 1 first strand synthesis 50 min, 42 ° C 2 Inactivation 15 min, 70 C PCR batch each for CfWT and Cfε (master mix for each 4x 50 µl batches) Approaches: (i) cdna from CfWT (ii) cdna from Cfε (iii) water control are additionally carried out with the primer pair X013 / X008 four independent reactions with 1 µl pgfruε [GK14] 50 ng / µl (provided by the assistant) are set up: 10

13 the 4 reactions are used to produce the radioactive probe for the Southern blot analysis described later ε-frustulin-gene β-tubulin-gene RT-PCR mixture 2 µl RT-PCR mixture 2 µl 5x PCR buffer 10 µl 5x PCR buffer 10 µl 2 mm dntps 5 µl 2 mm dntps 5 µl 10 pmol / µl X013 2 µl 10 pmol / µl TUB18 2 µl 10 pmol / µl X008 2 µl 10 pmol / µl TUB28 2 µl H2O 28.5 µl H2O 28.5 µl Taq Pol. 0.5 µl Taq Pol. Hold 0.5 µl samples on ice until the thermoblock of the PCR machine has reached 94 C Place sample vessels in the block Step 1 denaturation 2 min, 94 C 2 denaturation 30 sec, 94 C 3 annealing 30 sec, 58 C 4 extension 45 sec , 72 C 5 cycles go 40x back to 2 6 extension 7 min, 72 C 7 storage 4 C 1. 1/10 of the PCR reactions are analyzed in a 1% agarose gel 2. expected size of the PCR products: o X013 / X008: cdna 412 bp (genomic DNA: 661 bp) o TUB18 / TUB28: cdna 868 bp (genomic DNA: 1327 bp!) Isolation and phosphorylation of the PCR product X013 / X ethanol precipitation of the PCR mixture (90 µl) 2nd pellet dry (37 C) 3. dissolve in 10 µl TE buffer, pipette together on ice + 2 µl 10x T4-DNA-Pol buffer + 1 µl 2 mm dntp - mix + 5 U T4 DNA polymerase + H 2 O ad 20 µl 12 C / 15 min, inactivate immediately on ice: 70 C / 30 min 4. Electrophoresis in 1% agarose gel 5. Cut out the band (only use 366 nm UV lamp for detection!) 6. Using the Qiaex II gel extraction kit (5 µl Eq asmilch) 7. Elution with 10 µl H 2 O + 1 µl 10x T4-PNK buffer + 1 µl 10 mm ATP + 0.5 µl T4 polynucleotide kinase (2U) 1 h / 37 C 10 min / 70 C µl of the mixture analyze by electrophoresis in 1% agarose and estimate the amount. Store the rest at -20 C or use for ligation Isolate puc 18 and prepare for ligation (according to Birnboim and Doly) Plasmid preparation on a midi scale ml ÜN culture DH5a puc centrifuge at 4000 rpm, discard the supernatant 3. Pellet in Take up 1.5 ml LI, put on ice for 5 min. 4. Add 3 ml L II, invert; do not shake !!, 10 min on ice (no longer); Solution should become clear 5. Add 2.25 ml L III, invert, 10 min on ice; white flakes should appear centrifuge at 4 C for min 11

14 7. Precipitate the supernatant with 0.6 volumes of isopropanol; Centrifuge for 10 min at rpm and 4 C 8. Discard the supernatant, dissolve the pellet in 800 µl TE 9. Add 800 µl 5 M LiCl, centrifuge on ice for 10 min at 4 C, distribute the supernatant between two Eppendorf reaction vessels (2x 800 µl) 11. Precipitation with 2x 800 µl isopropanol, washing the pellets with 70% ethanol 12. Pick up the pellets in 250 µl TE, combine (now a total of 500 µl) 13. Add 2 µl RNase (10 µg / µl), Incubation for 30 min at 37 ° C. 14. Extract one after the other with 1 volume each of phenol, phenol / chloroform / isoamyl alcohol (25/24/1), chloroform / isoamyl alcohol (24/1) 15. Add 1/10 volume of 3 M sodium acetate ph 5.2, precipitation with 1 volume of isopropanol, washing with 70% ethanol 16. Absorb the pellet in 100 µl TE or water, remove 2.5 µl plasmid solution +2.5 µl TE +1 µl 6x DNA sample buffer (DNA-PP ) Quantity estimation in 1% agarose gel (1x TAE) Sma I digestion and phosphatase treatment of puc µg puc 18 are mixed with 40 U SamI in NEB4 buffer with BSA (0.1 mg / ml) in a total volume lumen of 100 µl digested at 25 C for 2 h after 1 h, 5 µl of the SmaI digest is removed and digested with 10 U XmnI in NEB4 buffer with BSA (0.1 mg / ml) in a total volume of 20 µl at 37 C for 1 h µl of the SmaI digest and the entire SmaI / XmnI digest, as well as 250 ng of undigested puc 18 are analyzed in an agarose gel (1%). 3. Remaining SmaI digestion: + 4 U CIP: 60 min / 37 C 4. Phenol / CHCl 3 extraction 5. Ethanol precipitation 6. Purification by electrophoresis in 1% agarose (only use 366 nm UV lamp for detection!) Gel elution after the Dialysis tube method (see Maniatis: with 1x TAE at 100 V) min are sufficient for complete elution of the DNA from the gel piece 2. Centrifugation 3. Supernatant Na acetate / ethanol precipitation 4. Dissolve pellet in 10 µl TE Eluate by means of agarose gel electrophoresis, ligation of the PCR product with puc18 and transformation L1 L2 PCR product (blunt end, 5 'phosphorylated) 50 ng ---- puc 18 (Sma I, CIP) 100 ng 100 ng + 10x T4-DNA Ligase buffer 1 µl 1 µl + 10 mm ATP 1 µl 1 µl + T4 DNA ligase 2 U 2 U + H2O to 10 µl to 10 µl RT, 4h or above. Incubate Transformation (see 2.4) Streak the transformation batches on AXI plates. Colony PCR (identification of clones that contain the PCR product) 9 white clones are "picked" as described under 2.5, streaked out, and analyzed by PCR. A water check. 12th

15 2x PCR mix (for eleven 50 µl reactions): 27.5 µl 2mM dntp-mix (provided) 110 µl 5x PCR buffer (provided) 27.5 µl 10 pmol / µl uni primer 27.5 µl 10 pmol / µl rev primer 5.5 µl Taq Pol. 77 µl H2O PCR program: keep samples on ice until the thermoblock of the PCR machine has reached 94 C Place sample vessels in the block Step 1 Denaturation 2 min, 94 C 2 Denaturation 30 sec, 94 C 3 Annealing 30 sec, 55 C 4 Extension 45 sec, 72 C 5 cycles go 40x back to 2 6 Extension 7 min, 72 C 7 Storage 4 C Analyze 10 µl of each batch on 1% agarose gel (expected product: approx. 500 bp). Plasmid preparation and restriction analysis 1. of 2 clones positive in the colony PCR, the plasmid is in each case made from 10 ml above sea level. Culture enriched (see 2.6) 2. Each 1 µg of plasmid DNA is digested with 5U EcoRI and 5U BamHI in EcoRI buffer with BSA (0.1 mg / ml) in 20 µl final volume at 37 C for 1 hour and digested in an agarose gel (1%) analyzed 3.2 Sequencing of the PCR product Sequencing reaction Denaturation of the miniprep DNA for the sequencing reaction: ~ 5 µg DNA (from miniprep with phenol / chloroform extraction and isopropanol precipitation) 4 µl 2 M NaOH ad 20 µl H 2 O 15 min, RT + 8 µl 5 M NH 4 acetate 100 µl ethanol p. a. (20 C) 15 min, 20 C centrifugation, 10 min, 4 C, Eppendorf centrifuge pellet with 70% ethanol p. a. wash, centrifuge, dry at 37 C Annealing: Pellet + 1 µl 2 pmol / µl primer 2 µl annealing buffer 10 µl H 2 O 15 min, 37 C (1) (1) 13

16 Polymerization (T7 DNA polymerase): (2) + 2 µl Labeling Mix 2 µl 1.5 U (2) / µl T7 DNA polymerase (2) 1 µl 0.2 M DTT 1 µl α 35 (2 ) S-dATP 5 min, RT Place 2.5 µl stop mixes [GATC] in cups, prepare (2) 4.5 µl reaction mixture for 5 min, 37 ° C (3) Add 5 µl stop solution for each Melt 95 C on ice, apply 2 µl per lane to the sequestering gel. Store at -20 C (1) pipetted on the wall, rinsed with the H 2 O (2) pipetted on the wall, centrifuged (3) pipetted into the cup lid, centrifuged sequencing gel (6% acrylamide gel) 33.6 g urea ultrapure (42% m / v) 12 ml acrylamide stock solution 16 ml 5 TBE ad 80 ml H2O (25.8 ml) filter through a 0.2 µm filter and degas (water jet pump) Base gel: 10 ml gel + 50 µl TEMED + 50 µl 25% by sequencing gel: 70 ml gel + 70 µl TEMED + 70 µl 25% by apparatus [BioRad SequiGen] treat the glass plate on the buffer tank with repellant (dichloro-dimethyl-silane in n-hexane) Buffer: 1 l 1 TBE electrophoresis flow: Reached approx. 30 min with 60 watts to a temperature of 55 ° C. Main run: 2 to 2.5 h with 50 watt gel work-up Gel on cover plate for 30 min in 2 l of 10% acetic acid techn. insert (fixation of the DNA, dissolve the urea), clap Whatman paper and cover with cling film at 80 C for 1.5 h in the gel dryer, dry X-ray film ÜN. apply and develop 3.3 Southern blot with restriction-digested genomic DNA from Cf WT and the transformant Cfε Isolation of the genomic DNA [Wizard Genomic DNA Purification Kit, Promega (see Appendix)] Algae digestion: 1. Approx. 500 ml CfWT or Cfε (each max. 5x10 8 cells) from fermenter (fermenter was prepared and inoculated 5 days beforehand; cell density should be approx. Cells / ml). Centrifuge cells (10 min 2000 g) and resuspend the pellet in a little supernatant and pour in N2, fl. freeze as pearls 2. under N2, fl. Grind in a mortar, transfer to a 2 ml cup (approx. 300 mg), incubate 2 ml Nucleic Lysis Solution (Art.No. A7941) min / 65 C in a water bath, mix occasionally 5. Cool to RT, pour on two 2- ml cups distribute RNase treatment: 1. + 1.5 µl each 10 mg / ml RNase A (DNase-free) min 37 C 14

17 Protein precipitation: 1. + 250 µl each Protein Precipitation Solution (item no.A7951), mix well min, 4 C centrifuge at maximum speed and remove the supernatants DNA precipitation: 1. Supernatants + 700 µl each isopropanol (0 , 7 vol.) 2. Mix until DNA visibly precipitates 3. Centrifuge 1 min, RT at maximum speed 4. Wash pellet with 600 µl 70% ethanol each 5. Centrifuge for 1 min, RT at highest speed 6. Centrifuge pellet in air min Dissolve DNA dry / analysis: 1. Add 100 µl TE buffer to the pellet and incubate for 15 min / 65 ° C in a water bath; Mix occasionally 2. Combine resuspended genomic DNA from CfWT or Cfε 3. Centrifuge for 5 min, RT at maximum speed. Supernatant: Store DNA solution at -20 C 4. Dilute 5 µl DNA solution in 100 µl H 2 O, photometric determination Check the DNA concentration and the ratio E 260 / E 280 (30 to 80 µg DNA, ratio 260nm / 280nm> 1.7) ng on agarose gel. Digestion of the genomic DNA from CfWT and Cfε 2 batches each with 15 µg DNA o with Incubate 30 U EcoRI in NEB EcoRI buffer in a total volume of 200 μl o with 30 U BamHI in NEB BamHI buffer in a total volume of 200 μl o 4 h / 37 C, remove 10 μl from each batch and check for 0.8% agarose gel if necessary Digestion worked, then ethanol precipitation (see 2.2.1) Dissolve the dried pellet in 30 µl TE. Production of the 35 S-labeled probe PCR amplification of a fruε gene fragment (per group): PCR reactions of 4 identical batches with the primer pair X013 / X008 and 1 µl pgfruε ([GK14] 50 ng / µl) as template (see 3.1.2) expected size of the PCR product: 671 bp 1. Combine PCR batches 2. Phenol / CHCl 3 extraction 3. EtOH precipitation 4. Dissolve the dried residue in 40 µl TE and distribute over 4 agarose gel pockets 5. Cut out the PCR product band (fruε gene fragment) (only use 366nm UV lamp for detection!) and elute using Qiaex (10 µl glass milk) (estimate of 1/10 of the eluate using agarose gel electrophoresis ). Radioactive labeling ng fruε gene fragment in 10 µl H2O µl random hexamers (OD260 / ml = 50) 3. 5 min / 95 C 4. 1 min / ice µl 10x Klenow-DNA-Pol.-Buffer min / RT µl mix from dgtp, dctp, dttp (each 1 mm) µl 0.2 M DTT µl α [35 S] -dATP (> 1000 Ci / mmol) (= 20 µci) µl Klenow DNA polymerase (1 U / µl) 15

18 11. 1h / 30 C µl datp (1 mm) min / 30 C min / 65 C µl STE µl in 5 ml scintering solution: radioactivity measurement 17. Purification via Sephadex G 50 Spin Column. Hettich centrifuge: 1500 rpm, 2 min (swollen gel material is placed in STE) 18. μl in 5 ml scintering solution: radioactivity measurement to determine the yield Transfer of the DNA to a nylon membrane and hybridization in a 0.8% agarose gel are the following samples applied: o each 7.5 µg of the concentrated digestion mixture (see 3.3.2) apply in a pocket of one o 0.75 µg undigested genomic DNA as reference o 1 µg of the DNA standard o positive control: 50 or 150 attomol of EcoR I / Nco I digested plamid pble_fruε (issued by the assistant) electrophoresis at 5V / cm until bromophenol blue front has reached 2/3 of the gel length DNA transfer to membrane according to capillary blot rule (modified according to Maniatis) gel in approx. 3 gel volumes of the following solutions for the specified Incubate times: 1. 20min 0.25M HCl (prepare fresh) 2. 2x 15min 0.5M NaOH / 1.5M NaCl 3. 2x 15min 1M NH 4 Ac transfer for at least 3-4h, possibly overnight (transfer solution: 1M NH 4 Ac) Blot setup (scheme table): Transfer solution 0.5kg 3x Whatman paper gel seal with Parafilm membrane 2xWhatman paper paper towels, approx. 3cm high Fixation of the DNA by UV cross-linking with nylon membrane ("Auto-crosslink" in the Stratalinker from Stratagene = 120 mj / cm 2). Prehybridization: 1. Carefully roll the membrane and transfer it into the hybridization tube. Add ml of hybridization buffer (provided) 3. Roll in the hybridization oven for at least 1 h / 65 C 4. Discard hybridization buffer Add ml of hybridization buffer Hybridization: 1. 5x 10 7 cpm of the 35 S-labeled probe add 120 μl of 10 mg / ml salmon sperm DNA 2. 5 min at 95 C, immediately put on ice, add S-labeled probe 4. o.n. / 65 C roll Washing the membrane: 1. Prewarm all washing buffers to 65 o C 2. Radioactive hybridization buffer: rad. Waste 3. Rinse the vessel 1x with 25 ml washing buffer 1 (3XSSC, 0.1% SDS): rad. Waste 16

19 4. Roll membrane 2x in 25 ml wash buffer 2 (0.3XSSC, 0.1% SDS) 15 min / 65 o C 5. Membrane 2x in 25 ml wash buffer 3 (0.1XSSC, 0.1% SDS) 15 min / 65 o C roll 6. membrane 2x in 25 ml wash buffer 4 each (0.1XSSC, 1.5% SDS) roll 15 min / 65 o C 7. dry membrane (RT) Autoradiography: radioactivity on the membrane is detected with the help of a phosphor imager system (LAS 1000) 3.4 Quantitative PCR with genomic DNA from CfWT and Cfε Quantitative PCR makes it possible to determine the number of copies of the transgene integrated into the C. fusiformis genome. For this purpose, a portion of the fruε gene of the diatom N. pelliculosa is amplified and the original amount of this target sequence is determined in relation to that of the fru-alpha-2 gene from C. fusiformis. The efficiency of the amplification with the primer pairs R16 / 17 and R14 / 15 is determined from the dilution series of the plasmid pble_FRUε and genomic DNA from WT cells. In addition, the absolute amount of integrated fruε-gen is calculated using the pble_fruε dilution series.all pipetting work is carried out in VKL10.10 with stuffed tips: Master mix, Sybr Green, HotStar Taq, primer at qpcr workstation DNA samples at the place one student per group pipettes the samples (genomic DNA in triplicate, water control) one student prepares a dilution series pble_fruε on and inoculated with this the qpcr reaction (in duplicate, water control). Dilutions of pble_fruε and genomic DNA A stock solution of pble_fruε (0.48pM; linearized with EcoRI) is diluted 1:20 in 10mM TRIS ph7.5 (24fM). A 1: 5 dilution series is made from this solution in 10mM TRIS pH7.5 so that the following concentrations are obtained: 4.8fM, 0.96fM, 0.19fM, 38aM. 1fM solutions of genomic DNA from WT and transformants should be prepared [Cf Genome ~ 95Mb; Average molecular weight of a base pair ~ 660] Approaches for genomic DNA samples from WT and transformants (primer pairs R14 / 15; R16 / R17) reactions 8 MM 9.67 77.36 HotStar Taq Qiagen 5u / ul 0.08 0.64 [1: ] SG Stock * 0.25 2 P # 1 [10µM] 0.40 3.2 P # 2 [10 µm] 0.40 3.2 Water 5.2 41.6 Total volume of the 16 µl are placed in 0.1ml reaction vessels presented and inoculated with 4 µl of the genomic DNA dilution (triplicates). (Don't forget: water control). In total, 6 reactions are set up for each genomic DNA sample (3x R14 / 15 mix + 3x R16 / 17 mix) and 2 reactions for the water controls (1x R14 / 15 mix + 1x R16 / 17 mix). 17th

20 3.4.3 Approaches for pble_fruε and WT genomic DNA dilution series (primer pairs R16 / R17 and R14 / 15) reactions 12 MM 9.67 116.04 HotStar Taq Qiagen 5u / ul 0.08 0.96 [1:] SG Stock * 0.25 3 P # 1 [10µM] 0.40 4.8 P # 2 [10 µm] 0.40 4.8 Water 5.2 62.4 Total volume of the 16 µl are placed in 0.1ml reaction vessels and marked with 4 µl of the respective DNA dilution inoculated (duplicates). (Don't forget: water control) a total of 11 reactions (2x each sample of the dilution series + 1x water sample) are set up Parameters for the RotorGene-3000 qpcr system from Corbett Research Cycle 95 c, 15 min 0 secs Cycling (40 repeats) Melt (60- 95 c), hold 45 secs on the 1st step, hold 5 secs on next steps, Melt A (FAM / Sybr) Cycle Point Step 95 c, hold 15 secs Step 60 c, hold 60 secs, acquiring to Cycling A (FAM / Sybr) Data analysis The data analysis includes: absolute quantification (absolute quantitation) comparative quantitation (comparative quantitation) melting curve analysis (Melting Curve) control in agarose gel of samples with identical melting curve, 5 µl of a qpcr reaction are analyzed in a 2% agarose gel electrophoresis. Expected amplicon sizes: R14 / 15: 188bp R16 / 17: 191bp 18

21 4 Appendix 4.1 Composition of the solutions used Solutions to be prepared (first day of the internship) For the following solutions, see Maniatis: TE (ph8.0; 50 ml, autoclave!) 3M NaAc ph5.2 (50 ml, autoclave!) 10xTAE (1 liter) STE (50 ml) 20x SSC (100 ml) 5x TBE (200ml) 2x YT medium (100 ml, autoclave!) LB medium (100 ml; autoclave!) SOB medium without MgCl 2 (100 ml, autoclave!) Production from LB / Amp plates: LB medium + 1.5% agar: autoclave 500 ml: allow to cool to approx. 55 C for 20 min + 0.5 ml 100 mg / ml ampicillin pour approx. 25 ml per plate (sterile bench) 30 min harden in the sterile bench and allow to dry (lid half open) store at 4 ° C. Production of AXI plates: as described for LB / Amp plates, with: 40 µg / ml X-gal 0.5 mm IPTG ASW medium [1000 ml] Glycylglycine NaCl 1M CaCl 2 2M MgCl 2 2M MgSO 4 3M KCl 1M KNO 3 100mM Na 2 SiO xSE solution 1mg / ml Thiamine H 2 O ad 1l with 1M NaOH ad ph 8.0 autoclave after cooling add 2 ml 100 mm K 2 HPO mg 23, 4 g 7.5 ml 10 ml 10 ml 2 ml 1.5 ml 2 ml 1 ml 0.5 ml 1000xSE solution [1000 ml] H 3 BO 3 1.14 g Na 2 EDTA 2H 2 O 6.05 g ZnCl mg CuCl 2 H 2 O 270 mg Na 2 MoO 4 2H 2 O 250 mg CoCl 2 6H 2 O 420 mg FeCl 2 4H 2 O 970 mg MnCl 2 4H 2 O 360 mg 19

22 1M Tris HCl ph 8.0 (50 ml) store at RT 0.5 M EDTA ph 8.0 (100 ml) store at RT H2O (50 ml, autoclave) at RT store 100 mg / ml ampicillin (1 ml, sterile filter) in media for bacterial cultures (LB / Amp or 2xYT / Amp) diluted 1: 1000 (final conc .: 100 µg / ml) store at 20 C 1M DTT (1 ml, sterile filtering) prepare 200 mm DTT dilution both store at 20 C 50 mm Tris 50 mm glucose 15 mm EDTA 5 mg / ml lysozyme from chicken egg white pH 8.0 LI (25 ml) 50 mm Tris 50 mm glucose 15 mm EDTA 5 mg / ml lysozyme from chicken egg white pH 8.0 L II (25 ml) 200 mm NaOH Store 1% SDS at RT L III (25ml) adjust 3M KAc with HAc to pH 4.8 (see Maniatis!) Store at 4 C H buffer (1ml) 100 mm Tris HCl pH 7.5 66 mm MgCl2 600 mm NaCl store at 20 ° C Tfb I (50ml, sterile filtering) 30 mm KAc 50 mm MnCl2 100 mm KCl 15% glycerine pH 5.8 with HAc store at 4 ° C Tfb II (50ml, sterile filtering) 10 mm Mops / NaOH pH 7.0 75mM CaCl2 10 mm KCl 15% glycerine stored at 4 C. Store 2.5 M CaCl 2 (1ml, sterile filter) at RT 20

23 DNA lysis buffer (1 ml) 2 M NaCl 0.1 M NaOH 0.5% SDS store at RT 2 M MgCl 2 (10 ml, autoclave) store at RT 1 M glucose (1 ml, sterile filtration) store at RT 10 M LiCl (autoclaved with DEPC) Water (autoclaved with DEPC) 1M ammonium acetate 10% SDS Issued solutions: 10x NEB buffer 100 mm Tris Ac ph 7.5 100 mm MgAc 500 mm KAc 5x PCR buffer 250 mm Tris HCl ph 8.8 250 mm NaCl 12.5 mm MgCl 2 10 mm DTT 10x T4-PNK buffer 500 mm Tris HCl ph 7.5 100 mm MgCl 2 50 mm DTT 10x T4 DNA polymerase buffer 670 mm Tris HCl ph 8.8 67 mm MgCl mm β-mercaptoethanol 166 mm (NH 4) 2 SO 4 67 µm EDTA 1.6 µg / ml BSA 10x T4-DNA-Ligase buffer 200 mm Tris HCl pH 7.5 50 mm MgCl 2 50 mm DTT 5x First-Strand Buffer 250 mm Tris HCl ph 8.3 375 mm KCl 15 mm MgCl 2 Fragmented salmon sperm DNA (10mg / ml) 6x DNA sample buffer (for agarose gels) 30% glycerine 100 mm EDTA ph 8.0 0.125% bromophenol blue 0.125% xylene cyanol 21

24 100 mm spermidine, free base (1 ml, sterile-filtered) store at RT 1M IPTG (sterile-filtered) 20 mg / ml X-Gal (in DMF) Sequencing solutions: Annealing buffer (1 ml) 280 µl 1 M Tris HCl ph 7 , 5 100 µl 1 M MgCl µl 1 M NaCl 270 µl H 2 O Labeling Mix (1 ml) µm dbtp 2 µl 100 mm dgtp * 2 µl 100 mm dttp 2 µl 100 mm dctp 994 µl H 2 O µl of µl H 2 O 3 µm per dbtp stop solution (1 ml) 980 µl deion. Formamide 20 µl 0.5 M EDTA ph 8.0 2 mg Bromophenol Blue 2 mg Xylenxyanol Stop-Mix [GATC] (1 ml each) Buffer 400 µl 1 M Tris HCl ph 7.5 100 µl 1 M MgCl µl 1 M NaCl 2 Mix 6 µl 100 mm dgtp * 3 µl 100 mm datp 6 µl 100 mm dttp 6 µl 100 mm dctp 1979 µl H 2 O [G] 100 µl buffer 500 µl 2 Mix 3 µl 5 mm ddgtp 397 µl H 2 O [A] 100 µl buffer 500 µl 2 mix 1.5 µl 5 mm ddatp 398.5 µl H 2 O [T] like [G] but ddttp [C] like [G] but ddctp 40% acrylamide stock solution 38 g acrylamide 2 g bis -Acrylamide ad 100 ml H 2 O, dissolve under heating, overnight. Shake with 2% mixed bed ion exchanger at RT., filter through a 0.2 µm filter, store at 4 ° C. 22nd

25 4.2 DNA Marker Log DNA Ladder (kb) NEB, Catalog # N

26 4.

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