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Methods

1. Molecular Biology Methods

Isolation of Plasmid DNA from E.coli (miniprep)

Plasmid DNA from E. coli was isolated from overnight cultures using the DNA extraction mini-prep kit (Qiagen).The principle of this method is the alkaline lysis of the bacterial cells followed by a selective immobilization of the plasmid DNA on a column, a couple of washing steps and the elution of the DNA.

Isolation of Genomic DNA from S.cerevisiae

The isolation of genomic DNA from S.cerevisiae was done using a protocol by Lõoke et. al (2011). This method is based on the lysis of yeast cells with a LiOAc-SDS-solution followed by the precipitation of DNA with ethanol. For DNA isolation, 100 µl from a statonary overnight culture was pelleted and resuspended in 100 µl of 200 mM LiOAc 1 % SDS. Cells were incubated for 5 minutes at 70 °C. By adding 300 µl of 96 % ethanol and vortexing, the DNA was precipitated. After centrifugation for 3 minutes at 13400 rpm and throwing away the supernatant, the DNA was washed with 500 µl 70 % Ethanol an centrifuged again. After the removal of the supernatant, the DNA was resuspended in 100 µl 1x TE buffer. Remaining cell debris was centrifuged down and the supernatat was transferred into a new tube. in order to purify the genomic DNA for the following PCR, another precipitation step with isopropanol was done.

Determination of DNA Concentration

DNA concentration was measured using a NanoDrop Spectrophotometer by Thermo Scientific. The concentration was calculated after determination of the specific absorbance of DNA at 260 nm. Furthermore, the ratio of sample absorbance at 260 and 280 nm and at 260 an 230 nm were measured to specify the purity of the samples. A ratio of 260/280 of ~1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate contemination with protein, phenol or other substances that absorb strongly at or near 280 nm. The ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purityad is often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2.

Agarose Gel-Electrophoresis

To separate double-stranded DNA fragments by length, agarose gel-electrophoresis using ethidium bromide as a nucleic acid stain was applied (Sambrook et al., 1989). This method was used for the restriction analysis of plasmids (analytical gel-electrophoresis) as well as for the isolation of DNA fragments (preparative gel-electrophoresis). After preparative gel-electrophoresis, the bands were cut out and purified using a Qiagen Gel extraction kit.

Polymerase Chain Reaction (PCR)

For the selective amplification of a desired DNA fragment (for example from a plasmid), the polymerase chain reaction (PCR) was used. Specially designed primers define the desired target sequence. These primers serve as starting points for the polymerase which then extends the newly synthesized DNA strand. The PCR reaction is divided in three steps which are repeated up to 30 times. Firstly, the DNA template strand is heat-denatured at 95 °C to produce single-stranded DNA. Secondly, the temperature of the reaction batch is lowered to 55 – 60 °C to allow the primers to bind. Thirdly, the temperature is raised to 72 °C. This enables the DNA polymerase to synthesize the other DNA strand.

Special PCR methods that were used include colony and genomic PCR.

purification of PCR products

PCR products were purified using the PCR purification kit by Qiagen.

Colony PCR

A method to allow for higher throughput of clone screenings. One colony each from respective agar plate needed to be picked with a sterile toothpick or pipet tip. Some of the cells were smeared onto the wall of the PCR tube. Subsequently the toothpick was put into a cell culture tube with LB-medium and suitable antibiotic. Colony PCR was performed using OneTaq Hot Start DNA Polymerase (Qiagen) after following temperature scheme:

Initial denaturation	94 °C	10 min
30 cycles	95 °C	30 s
	59 °C	30 s
	68 °C	1 min 55 sec
Final extension	68 °C	5 min
Hold	4 °C

Dephosphorylation of DNA

To avoid religation of the insert and enhance ligation rate Dephosphorylation of digested PCR products via Shrimp Alkaline Phosphatase (Fermaentas) was made. Befor Dephosphorylation procedure could be performed, the PCR Purification Kit of Qiagen were used for purifying digest solution of restriction enzymes and buffer. Afterwards 1 µg sample DNA was mixed with 10% of 10x SAP Buffer and 1 unit SAP. The mixture incubated at 37°C for 30 min. Inactivation occurred at 65°C for 15 min.

DNA restriction enzyme digest

For the preparation of DNA fragments and the restriction analysis of plasmid DNA, the DNA was cut using restriction endonucleases. For restriction digestion, buffer and DNA concentrations were used according to the suggestions by the manufacturer.

ligation / cycled ligation

After digestion with an restriction enzyme, plasmid fragments can be inserted into a vector (which was cut with the same restriction enzymes) by ligation. Ligases, such as the enzyme T4 ligase which was used in our ligations, connect complementary overhangs of fragments by catalyzing the formation of the bond between the 5'phosphoryl group and the 3' hydroxyl group.

oligohybridization of single-stranded DNA

Oligohybridization of oligo-nucleotides was for example used to create a mini MCS for cloning RFC10 compatible parts between promoter and terminator. This was achieved by using complementary oligo-nucleotides that contained the desired sequence with specific overhangs for cloning. For oligohybridization, 25 ml of 100 mM of forward and reverse oligos were put together in one tube and heated to 90 °C for 5 min. the samles were then slowly cooled to room temperature in a styrofoam box overnight.

Site-Directed Mutagenesis

Site-Directed Mutagenesis was used to mutate a specific base inside a sequence. Therefore, specific primers, which bind at the same site and contain a mismatch at the specific base are required. The original base pair that has to be replaed is replaced by the mismatch. The method works just as PCR by amplifying the desired product that contains the mismatch.Afterwards, the product ist digested with the restriction enzyme DpnI to destroy the plasmids strands which do not contain the desired base pair exchange.For the site directed mutagenesis, the QuikChange Site-Directed Mutagenesis Kit by Agilent Technologies was used.

sequencing of plasmid DNA

DNA constructs were sequenced by [http://eurofinsdna.com/ Eurofins mwg operon] using our own sequencing primers.

gene synthesis

Some of our genetic constructs were synthesized by GeneArt and iDT. For all our syntheses, the GeneArt® GeneOptimizer® was used for codon optimization in S.cerevisiae.

2. Protein Biochemical Methods

Protein expression in S.cerevisiae

Expression of our proteins was induced by transferring a overnight culture of our INVSc1 strain in glucose medium into a medium that contained galactose.In INVSc1, transcription from the GAL1 promoter is repressed in the presence of glucose. Transcription may be induced by removing glucose and adding galactose as a carbon source. Transferring cells from glucose- to galactose-containing medium causes the GAL1 promoter to become de-repressed and allows transcription to be induced.

Crude protein extraction from S.cerevisiae

To detect the recombinant protein (for example by western blot) cell lysates from our yeast transformants were prepared. Cell lysates were created using a special breaking buffer and acid-washed glass beads to break the cell wall.

SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE)

Firstly, glass plates were wiped with 70% ethanol and then assembled onto a setting rig. No rubber spacers were added as they were already fixed to the glass plates. A 7% resolving gel was made first as follows:

5 ml sterile water
2.5 ml Tris (1.5 M pH 8.8) (BDH)
100 μl SDS (10%) (BDH)
2.33 ml acrylamide:bisacrylamide (37:1, 30%) (Sigma)
100 μl ammonium persulphate (APS) (10%) (Sigma)
10 μl N,N,N’,N’-tetramethylethylenediamine (TEMED) (Sigma).

The tube was mixed thoroughly and 7 ml added to the setting rig between the glass plates, covered with water ethanol and left to set for about 30 minutes. Then the water was poured off and a stacking gel was prepared as follows:

5 ml sterile water
2.5 ml Tris (0.5 M pH 6.8)
100 μl SDS (10%)
1 ml acrylamide:bisacrylamide (30%)
100 μl APS (10%)
10 μl TEMED.

Again all ingredients were mixed by inversion and then 1-2 ml applied on top of the already set resolving gel and a comb put in. Once the gel was set, the comb was taken out and the wells cleaned out with sterile water to ensure the removal of all acrylamide. The set gels were removed from the setting rig and placed in the running rig. 1x running buffer was poured into the rig ensuring the plates were covered.

Western Blot

After SDS-PAGE gels were transferred to a nitrocellulose membrane in transfer buffer (20 ml 5x SDS buffer, 20 ml methanol, 60 ml H2O)at 500 mA for 1h. Membranes were washed 3x 15 min in PBS-T0.1 (PBS + 0,1 % v/v Tween 20)and then blocked with 3 % BSA for one hour. For antibody detection, membranes were washed 3x 15 min in PBS-T0.1 and then incubated in detection solution containing the antibody straptavidin-AP (1:4000 in PBS-T0.1) for one hour. The Western blot was then washed 2x 10 min in PBS-T0.1 and then 2x 10 min in PBS. Afterwards, the developing solution (15 ml alkaline phosphatase buffer, 45 µl BCIP (50 mg/ml in DMF) and 7,5 µl NBT (75 mg/ml in 70 % DMF) was added. After the appearance of bands, the blot was washed and stored in water.

Enzyme Assay and Extraction of Limonenesynthase

To test functionality of purified Limonenesynthase we used an optimized protocol of [http://www.ncbi.nlm.nih.gov/pubmed/17662687 Landmann et al, 2007] . The Enzyme Assay was carried out in a total volume of 500 µl containing buffer (25 mM Tris-Cl, pH 7.5, 5 % glycerol, 1 mM DTT) supplemented with cofactors (10 mM MgCl2, 1 mg/ml BSA) with successive addition of 50 µM substrate (geranyl pyrophosphate, dissolved in DMSO) and 10 µg purified recombinant enzyme (extracted limonene synthase, after Purification). The mixture was gently overlaid with 1 ml pentane and incubated at room temperature for 15 minutes. The reaction was stopped by vigorous mixing and centrifugation (5 min, 5000g) to separate phases. The solvent phase (upper phase) was attached to a pasteur pipette containing glass wool with Sodiumsulfate for drying the solvent phase. Afterwards the combined extracts were reduced to approximately 300 µl under a stream of nitrogen. The pentane extracts were analyzed with gas chromatography-mass spectrometry to identify the enzymatically synthesized products. An aliquot of each sample (0.5 µl) was injected into "5890 Series II GC" coupled to a "Finnigan Mat 55 S MS".

Headspace GC-MS of Limonene

Because Limonene is an secretory protein [http://www.ncbi.nlm.nih.gov/pubmed/21310602 [Misawa, 2011]] we expected an arbitrarily amount of Limonene outside the cells. To check this predication we detected Limonene via Headspace GC-MS in the yeast cell culture supernatant. Therefore the preparatory cell culture was induced with galactose after 24 hours (see 'Protein expression in S.cerevisiae). For further 24 hours the protein expression proceeded. Afterwards the SPME needle was injected into the headspace and incubated for 30 min at 45 °C before injection into GC.

3. Microbiological Methods

cultivation of E.coli

E.coli strain XL1-Blue was cultivated in LB-medium (lysogeny broth) and LB plates. for the preparation of 1 liter of LB, dissolve the following and autoclave:

10 g tryptone
5 g yeast extract
10 g NaCl

for making plates, add 15 g bacto-agar before autoclaving

cultivation of S.cerevisiae

S.cerevisiae strain INVSc1 was cultivated using the following media and plates:

SC-Uracil minimal Medium and plates for 10x 50 ml amino acid aliquots: Dissolve the following reagents in 500 ml deionized water:

1 g of adenine, arginine, cysteine, leucine, lysine, threonine, tryptophan and
0.5 g of aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine.
Autoclave at 15 psi, 121°C for 20 minutes.
Make 50 ml aliquots, store at -22°C.

SC-U medium + Glucose (minimal medium) or Galactose (induction medium):

Add 6.7 g Yeast Nitrogen Base and 850 ml deionized water to the 50 ml amino acid aliquot. Autoclave. Add 100 ml autoclaved/filter sterilized 20% glucose (minimal medium) or 20% galactose (induction medium), store at room temperature.

SC-plates + Glucose:

Add 6.7 g Yeast Nitrogen Base and 850 ml ELGA and 20 g agar to the 50 ml aliquot. Autoclave.Add 100 ml autoclaved/filter sterilized 20% glucose.5. Pour plates and allow to harden. Invert the plates and store at 4°C. Plates are stable for 6 months.

heat shock transformation of E.coli with plasmid DNA

Before transformation, CaCl₂ competent cells were created after Cohen et al., 1972. For the creation of competent cells, 50 ml LB medium were inoculated with an overnight culture of the used ‘’E.coli’’ strain and incubated at 37 °C, 180 rpm. After an OD ₅₅₀ of 0,5 was reached, the culture was centrifuged for 4 minutes at 5000 g for 10 minutes. The pellet was then resuspended in in 40 ml pre-chilled in 0,1 M MgCl₂ solution, centrifuged again and then again resuspended in 20 ml of pre-chilled 0,05 M CaCl₂ solution. After 30 minutes of incubation on ice, the cells were centrifuged again and resuspenden in 2 ml 0,05 M CaCl₂ solution, 15 % v/v glycerol. The competent cells were aliquoted and stored an – 80 °C. For the transformation, 100 µl competent cells and 1 ng plamid or 5 µg of a ligation product were mixed and incubated for 30 minutes on ice. Afterwards, the cells were heat shocked at 37 °C for 5 minutes, then mixed with 2 ml LB medium and incubated at 180 rpm and 37 °C for 30-45 minutes. The transformed cells were then plated on LB medium containing an antibiotic.

transformation of S.cerevisiae

Yeast cells were transformed using the S. c. EasyComp Transformation Kit by Invitrogen. Firstly, cells were grown in YPD medium to mid-log phase. Then, cells were pelleted and washed with a washing solution. Afterwards, cells were pelleted again and washed with Lithium cation solution in order to make the cells competent. Finally, the cells were aliquoted and stored at - 80 °C. For yeast transformation, frozen competent cells were mixed with up to 5 mg of plasmid DNA and transformation solution. The transformation batch was mixed by vortexing and incubated at 30°C to induce uptake of DNA for one hour. then the transformed cells were plated on selective plates and grwon for 2-4 days at 30 °C.

genome integration

4. Chemical Methods

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder by methanolysis was done according to a protocol by Jim Tepperman (http://openwetware.org/images/2/27/Y2H_documentation.pdf). 50 g Spirulina powder were suspended in 1.5 liter H2O (30 ml/g) in a flask covered with aluminium foil. The mixture was stirred for 10 minutes and then centrifuged (GS3) at 8000 RPM at 4ºC for 1 hr. The supernatant was decanted and 15 g TCA (final concentration of 1% (w/v)) was added. The mixture was stirred for 1 hr in the dark (aluminum foil cover) at 4º C and then centrifuged at 8000 RPM at 4º C for 10 min. The supernatant was discarded. The pellets were washed on ice several times with MeOH until the formerly green supernatant turned blue. The pellets were stored at 20 °C overnight, wrapped in aluminium foil. Since the free chromophore is very susceptible to photobleaching, the following steps were performed under green ‘safelight’ conditions in a darkroom. The next day, all pellets were resuspended in a final volume of 500 ml MeOH. Then the suspension was heated in a water bath at 70– 75 ºC with a condensing coil cooled with tap water for 5-8 hrs. The suspension was then centrifuged at 8000 RPM, GS3 rotor, 4º C for 20 min and the supernatant was filtered through miracloth. The filtered suspension and the pellets were stored at 20 °C overnight in the dark. The next day, a second methanolysis of the pellets from the first methanolysis was done. The two supernatans were pooled, the volume of MeOH was reduced to 50 ml using a rotary evaporator and then transferred separatory funnel where it was diluted with 100 ml water. The solution was mixed with 50 ml chloroform and shook to obtain the PCB through solvent extraction. The solvent extraction with chloroform was repeated until the aqueous phase was alsmost colorless. The chloroform was evaporated with a rotary evaporator and a stream of nitrogen gas was blown over the remaining PCB to remove residues of chloroform. The dried PCB as dissolved in DMSO, aliquoted and stored at -80 °C.

References:

[http://www.ncbi.nlm.nih.gov/pubmed/20010584 Pubmed: Prasher, 1995: Using GFP to see the light.(Review)]
[http://www.ncbi.nlm.nih.gov/pubmed/17662687 Pubmed: Landmann et al, 2007: Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia).]
[http://www.ncbi.nlm.nih.gov/pubmed/21310602 Pubmed: Misawa, 2011: Pathway engineering for functional isoprenoids.]

@@ Line 87: / Line 87: @@
 ====SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE)====
+Firstly, glass plates were wiped with 70% ethanol and then assembled onto a setting rig. No rubber spacers were added as they were already fixed to the glass plates.
+A 7% resolving gel was made first as follows:
+* 5 ml sterile water
+* 2.5 ml Tris (1.5 M pH 8.8) (BDH)
+* 100 μl SDS (10%) (BDH)
+* 2.33 ml acrylamide:bisacrylamide (37:1, 30%) (Sigma)
+* 100 μl ammonium persulphate (APS) (10%) (Sigma)
+* 10 μl N,N,N’,N’-tetramethylethylenediamine (TEMED) (Sigma).
+The tube was mixed thoroughly and 7 ml added to the setting rig between the glass plates, covered with water ethanol and left to set for about 30 minutes. Then the water was poured off and a  stacking gel was prepared as follows:
+* 5 ml sterile water
+* 2.5 ml Tris (0.5 M pH 6.8)
+* 100 μl SDS (10%)
+* 1 ml acrylamide:bisacrylamide (30%)
+* 100 μl APS (10%)
+* 10 μl TEMED.
+Again all ingredients were mixed by inversion and then 1-2 ml applied on top of the already set resolving gel and a comb put in. Once the gel was set, the comb was taken out and the wells cleaned out with sterile water to ensure the removal of all acrylamide.  The set gels were removed from the setting rig and placed in the running rig. 1x running buffer was poured into the rig ensuring the plates were covered.
 ====Western Blot====

Team:TU Munich/Notebook/Protocols