C. reinhardtii could be grown in liquid culture in Erlenmeyer flasks. In this thesis, 100ml Erlenmeyer flask with ~50ml culture medium was commonly used for growing Chlamydomonas. To prevent Chlamydomonas from precipitating or sticking together, the liquid culture in Erlenmeyer flasks were shaken or stirred continuously. The shaking speed was around 120rpm and stirring speed was around 200rpm. According to different requirement, different light intensities were applied to Chlamydomonas cultures. High light condition was ~9-10W/m², middle light condition was ~4W/m² and low light condition was less than 1W/m². The growing tempeature was 25ºC.

For screening large numbers of transformants, 96-well plates and 24-well plates were used for growing single Chlamydomonas clones. Those plates were transparent and made of plastic. They were placed under middle light condition (~4W/m²) and were kept still.

For light gradient experiment, a home made light filter was used to create a light intensity gradient (20W/m², 14.2W/m², 11W/m², 9.4W/m², 5.2W/m², 3W/m², 1.6W/m², 0.71W/m², 230μW/m², 160μW/m², 95μW/m²). 24 well plates were used for growing cells. They were kept still and were blown with an electric fan to get rid of the heat caused by the strong light intensity.

Both HSA (high salt acetate) and TAP (Tris-Acetate-Phosphate) were used as media for vegetatively growing Chlamydomonas. For strain 806 and cw2, HSAS was used. For strain cw15 arg- A or CC48 arg-, TAPA, TAPAYZ, TAPATYZ or TAPAP was used. For wild type strain, CC124mt(-), CC125mt(+), CC620mt(+), CC621mt(-) and bald strain CC477, CC478 and CC479, TAP or HSA was chosen as growing media. For dark growing culture, TAPTY was used. For CC32pab1mt(+), TAPP, TAPY, TAPYP or TAPPP was used.

TAP (Tris-Acetate-Phosphate)

NH₄Cl

7.5 mM

MgSO₄

0.4 mM

CaCl₂

0.34 mM

K₂HPO₄

0.54 mM

KH₂PO₄

0.46 mM

NaAc

5 mM

Tris/Cl pH 7.0

20 mM

Hunter's trace elements

0.1%

Hunter's trace elements

ZnSO₄ · 7H₂O

22 g

H₃BO₃

11.4 g

MnCl₂ · 4H₂O

5.06 g

FeSO₄ · 7H₂O

4.99 g

CoCl₂ · 6H₂O

1.61 g

CuSO₄ · 5H₂O

1.57 g

(NH₄)₆Mo₇O₂₄ · 2H₂O

1.1 g

EDTA

50 g

Dissolve in 1 liter water

TAPA(Tris-Actate-Phosphate-Arginine)

TAP medium with 50mg/l L-arginine

TAPAYZ(Tris-Actate-Phosphate-Arginine-Yeast extract-Zeocin)

TAPA medium with 10mg/l zeocin and 0.3%Yeast extract

TAPATYZ(Tris-Actate-Phosphate-Arginine-Trypton-Yeast extract-Zeocin)

TAPAYZ medium with 0.2% Bacto-Tryptone

TAPAP(Tris-Actate-Phosphate-Arginine-Paromomycine)

TAPA medium with 10mg/l paromomycine

TAPTY(Tris-Actate-Phosphate-Arginine-Trypton-Yeast extract)

TAP medium with 0.2% Bacto-Tryptone and 0.3%Yeast extract

TAPY( Tris-Actate-Phosphate-Yeast extract)

TAP medium with 0.3% Yeast extract

TAPYP( Tris-Actate-Phosphate-Yeast extract-Paromomycine)

TAPY medium with 10mg/l Paromomycine

TAPPab( Tris-Acetate-Phosphate-PAB)

TAP medium with 50mg/l PAB (p-aminobenzoic acid)

TAPP(Tris-Acetate-Phosphate-Paromomycine)

TAP medium with 10mg/l paromomycine

TAPPP( Tris-Acetate-Phosphate-PAB-Paromomycine)

TAPP medium with 10mg/l Paromomycine

HSA( High Salt Acetate)

NH₄Cl

9.3 mM

MgSO₄

0.81 mM

CaCl₂

0.1 mM

K₂HPO₄

6.2 mM

KH₂PO₄

6.8 mM

NaAc

15 mM

Hunter's trace elements

0.1% (v/v)

HASS(High Salt Acetate Sorbitol)

HAS medium with 250mM Sorbitol

Standard bacteriological petri plates(150×15mm) were used for growing Chlamydomonas transformants. 1.5% agar was added to the media mentioned above. Those plates are placed in 25ºC with 4W/m² light.

For keeping strains, agar slants in test tubes were used. Those agar slants contain 1.5% to 2% agar and TAPTY medium. Screw-capped test tube were used. Stock culture were kept in 18ºC under low light (<1W/m²).

Gametes were generated by resuspending vegetative cells in nitrogen minimal medium (NMM). Vegetative cells were collect by centrifugation at 2,500 rpm× 5min. The pellet was resuspended in NMM and centrifugated at 2,500 rpm× 5min again. Then the cells were resuspended in NMM at a density of ~1×10⁷ cells/ml and were placed under continuous light for 48 hours. To prepare pre-gametes, vegetative cells were collected and washed the same way as above. The the pellet was resuspended in NMM at a density of ~1×10⁷ cells/ml and was incubated in darkness continuously for 48 hours. Gametes could also be prepared from pregamete by incubating pregametes under continuous illumination for 2 hours.

NMM (Nitrogen minimal medium)

MgSO₄

0.81 mM

CaCl₂

0.1 mM

K₂HPO₄

6.2 mM

KH₂PO₄

6.8 mM

Cells grown in 2-liter culture were harvested by centrifugation (2,500rpm×5min) and resuspended in 100ml 10mM Tris buffer, pH7.8. Then the cells were collected at 2,500rpm for 5 min again and resuspended in 100ml 10mM Tris buffer, pH7.8. The cells were centrifugated again and the pellet was resuspended in 100ml 7% sucrose-10mM Tris buffer, pH7.8, at 4ºC. All the steps after this were carried out at 4C and it was essential to work quickly. Sedimented cells left in a pellet rapidly lysed, releasing their internal constituents and contaminating the final preparation of flagella.

The suspension of cells in 7% sucrose-Tris was vigorously stirred with a magnetic stirrer and 910µl 0.5N acetic acid was quickly added to rapidly decrease pH to 4.5. After 90 sec, 90-100% of the cells deflagellated. 1080µl 0.5N KOH was then added to the suspension to raise pH to 7.8.

For separation of the flagella, 15-ml aliquots were transferred to chilled 50-ml conical polycarbonate centrifuge tubes. 20ml of 25% sucrose-10mM Tris buffer was then placed under the 15ml aliquot of cell suspension. The tubes were centrifuged at 2000g for 11 min. The flagella were left in the 7% sucrose-10mM Tris buffer layer. The 7% sucrose-10mM Tris buffer layer was then collect and merged.

To remove any remaining cell bodies, 20ml portion of flagella suspensions were underlayered with 4 ml 25% sucrose-10mM Tris buffer as above and centrifuged for 9 min at 1800g. The supernatant was collected and centrifuged at 27,000 g for 20 min. The sedimented flagella were then obtained.

To obtain soluble fraction of flagella, ice-cold F buffer(pH7.4) was used to resuspend flagella pellet. The buffer volume was also around 1.5 times of the initial packed cell volume. F buffer was composed of 30mM HEPES, 5mM MgSO₄, 1mM dithiothreitol, 0.5mM Na₂EDTA, 25mM KCl, 0.5% polyethylene glycol (20,000 Da). It was FN buffer without detergent. 1mM phenylmethysulfonyl chloride (PMSF) was added to minimize proteolysis. Repeated freezing in liquid nitrogen and thawing in 37°C incubator were applied to the resuspension. Then the resuspension was centrifuged at 100,000g for 40 min. The supernatant was considered as the soluble fraction of flagella. The pellet was considered as the insoluble fraction of flagella.

Wild-type strain CC124mt(-) and CC125mt(+) were pre-grown in 500-ml liquid phototrophic culture for 2 days to a final cell density of ~7×10⁶ cells/ml. Cells were then harvested by centrifugation at ~2000rpm for 5 min. The pellet was washed once with NMM and then resuspend in NMM at a density of 2×10⁶ cells/ml. The cultures were illuminated with continuous light for 24 hours. Gametes were then harvested and resuspended at a density of ~4×10⁷ cells/ml in NMM. The two mating types were mixed and mated for 1 hour.

After mating, cells were collected by centrifugation at 3000g for 5 minutes. The supernatant fraction was collected and spun at 18,000g for 10min. Then the supernatant was lyophilized and resuspended in a small volume of NMM for use.

In this thesis, one metabolic deficient strain CC32pab1mt(+) with promomycin resistance was chosen as one mating partner and one wild type strain CC124mt(-) was chosen as another mating partner.

Pre-grown culture of both mating types were grown to mid-log phase under middle light condition (~4W/m²) in TAPTY medium. Cell density was measured. About 5×10⁶ cells of CC124mt(-) were inoculated in fresh TAP medium. Around 5×10⁶ cells of CC32pab1mt(+) transformants C4 and G5 were inoculated in fresh TAPPab medium. All the cultures were incubated in low light condition (~1W/m²) at 25°C for 5-7 days until cell density reached 2-3×10⁶ cells/ml.

The cells were then harvested and the cell numbers of different strains were counted. The pellet was washed once with NMM and resuspended in NMM at 1×10 ⁷ cells/ml. The cell suspension was kept in low light condition for 2 days (48 hours) at 25°C.

The cells were harvested again and cell number of different strain was counted. 5×10⁶ cells of C4 and 5×10⁶ cells of CC124mt- was mixed, and 5×10⁶ cells of control strain G5 and 5×10⁶ cells of CC124mt- was mixed. Those mixtures were kept in 2 ml sterile cups. The cells were spun at 1000g for 4 min and the volume was adjusted to 1ml in each mating reaction. The pellet was resuspended by vortex. Those cups were then placed in 25°C baker with light (~8W/m²).

After one hour, the cell mixtures were centrifuged at 1000g for 4 min again and 500µl of NMM was discarded. The pellets were resuspended again and were plated on TAPP agar plates. Those plates were kept in high light condition (~9W/m²), middle light condition (~4W/m²) or low light condition (<1W/m²) at 25°C. Every 7 days, cell number on each plate was checked and compared.

Cells were grown to a density of 5×10⁶ cells/ml and harvested by centrifugation at 2,500 rpm for 5 min. The pellet was then resuspended in ME buffer and sonicated 15times in ice bath. The homogenate was centrifuged at 13,000g for 15min. The pellet was composed of large organelles. The microsomal fraction was pelleted by centrifugation at 120,000g for 40 min. The supernatant after this centrifugation was regarded as cytoplasmic fraction.

ME buffer

MOPS

10mM

EDTA

1mM

Adjust pH to 6.8

Diatom cells could be grown in liquid culture in Erlenmeyer flasks. In this thesis, 250ml Erlenmeyer flask with ~100ml culture medium was commonly used for growing diatom. The cells were kept in 18°C room with continuous light illumination (~10W/m²).

Since diatom cells had a strong tendency to stick together and form big clump of cells, cell culture should be shaken vigorously. The cultures were normally shaken at 300rpm.

ASW medium (Artificial Sea Water medium) was used to grow diatom. The diatom strain could either be kept in liquid culture or agar slant (2%). In either case, culture should be kept in dim light (<1W/m²). When strain was kept in liquid, test tube filled with 5-7cm ASW medium was used. The medium should be renewed every four weeks. For the strains kept on agar slant, the strains should be inoculated onto fresh slant every two months.

ASW-Medium

Glycylglycine

600mg

NaCl

23.4g

1M CaCl₂

7.5ml

2M MgSO₄

10ml

2M MgCl₂

10ml

3M KCl

2 ml

0.5M KNO₃

3 ml

100mM Na₂SiO₃

2 ml

1000 ×SE solution

1 ml

1mg/ml Thiamin

0.5ml

Dissolve in 1 liter H2O and adjust to pH8.0 with 1M NaOH. Autoclave the medium. After cooling down to room temperature, 2ml of 100mM K ₂ PO ₄ should be added.

1000 ×SE solution

H₃BO₃

1.14g

Na₂EDTA 2H₂O

6.05g

ZnCl₂

620mg

CuCl₂ H₂O

270mg

Na₂MoO₄ · 2H₂O

250mg

CoCl₂ · 6H₂O

420mg

FeCl₂ · 4H₂O

970mg

MnCl₂ · 4H₂O

360mg

Dissolve in 1 Liter H₂O

The standard medium for E. coli growth was LB. For plasmid preparation, TB was used. In the liquid culture, the cells were grown at 37°C with shaking (250rpm). Transformants for E. coli were screened on LB agar plates with ampicillin (100µg/ml), the plates were kept in 37°C baker for clones growing.

LB

Bacto Trypton

10g

Bacto Yeast extract

5g

NaCl

10g

Dissolve in 1 Liter H ₂ O and autoclave.

TB

Media component

Bacto Trypton

12g

Bacto Yeast extract

24g

Re-distilled glycerol

4g

Dissolve in 900ml H₂O and autoclave.

Phosphate component

KH₂PO₄

2.3g

K₂HPO₄

12.5g

Dissolve in 100ml H₂O and autoclave. When the media and phosphate solution are cool, combine them to give 1 liter of TB.

A 2-3 mm diameter colony was picked from a freshly streaked SOB agar plate and inoculated in 250ml Erlenmeyer flask containing 50ml SOB medium (The cells were best streaked from a frozen stock). The culture was kept in 37°C baker overnight with continuously shaking (>180rpm).

On the next morning, the overnight culture was inoculated in 500ml Erlenmeyer flask containing 200ml SOB medium until the OD₅₇₈ reached around 0.05. The culture was then kept in 37°C baker for around 2-3 hours till OD₅₇₈ reached 0.3-0.4.

The cells were harvested by centrifugation at 2400 rpm for 7 min (4°C). The pellet was resuspended in 15ml Tfb I buffer(kept on ice before use). The incubation times varied for different strains. For DH5 α the incubation time should be 10 min while for BL21 the incubation time should be 30 min. The suspension was spun at 2,000 rpm for 5 min (4°C). The pellet was resuspended with 2ml TfbII buffer (kept on ice before use).

Aliquot of 110µl of the cell suspension was put into 500ml PCR tube and frozen quickly in liquid nitrogen. The cells were kept in -80°C freezer and could be used at any time.

SOB-Mg

Bacto tryptone

20g

Bacto yeast extract

5g

BaCl

10 ml of a 1M stock

KCl

2.5ml of a 1M stock

Dissolve in 1 liter DDW and autoclave.

MgCl ₂ +MgSO ₄ 2M stock

Combine 203g/l MgCl₂ · 6H₂O and 247g/l MgSO₄ · 7H₂O in DDW and sterilize by filtration through a pre-rinsed 0.2um filter unit.

SOB medium

SOB-Mg medium

1 liter

MgCl₂ + MgSO₄

10 ml of a 2M stock

Tfb I

KAc

30mM

MnCl₂ · 2H₂O

50mM

KCl

100mM

Glycerol

15%(v/v)

Use HAc to adjust pH to 5.8 and sterilize by filtration through a pre-rinsed 0.2um filter unit. The solution should be kept in 4°C.

Tfb II

MOPS

10mM

CaCl₂

75mM

KCl

10mM

Glycerol

15%(v/v)

Use NaOH to adjust pH to 7.0 and sterilize by filtration 987ewqthrough a pre-rinsed 0.2um filter unit. The solution should be kept in 4°C.

The tubes prepared from the step above was removed from the freezer and thawed on ice. DNA solution (volume < 10ul) was added. The tube was swirled gently to mix the DNA evenly with the cells. The tube was then incubated on ice for 10-30 min.

The cells were then treated with heat shock by placing the tube in a 42°C water-bath for 90 sec. The tube was chilled on ice for 1-2 min. 800µl of SOC medium was added in the tube and the tube was incubated at 37°C with moderate agitation for 30-60 min. The suspension was spread on SOB agar plate containing appropriate antibiotics. The plate was incubated at 37°C overnight. Colonies would appear in 12-16 hours.

SOC medium

SOB-Mg medium

1 litre

MgCl₂ + MgSO₄

10 ml of a 2 M stock

Glucose

10 ml of a 2 M stock

mRNA of target protein was prepared by RNA synthesis kit (Ambion). 10 ng of mRNA was injected into one mature oocyte. Those injected oocytes were kept in 18°C with constant agitation.

Three days later, the injected oocytes were harvested, each in one 500µl PCR tube. 50µl of HbA buffer was added. The oocytes were broken by pipetting. The tubes were centrifugated at 200g for 1 min and the supernatant was transferred to another clean tube. 2µl of supernatant was used for dot blot. The supernatant of those oocytes which expressed target protein was kept in -20°C for use.

HbA buffer

Tris HCl (pH7.6)

20mM

MgCl₂

5mM

NaH₂PO4

5mM

EDTA

1mM

Saccharose

80mM

The Isolation kit was made by Promega and the protocol was changed a little in this thesis. It is described as below:

The cells were grown to a density of 2 × 10⁶ - 1 × 10⁷ cells/ml and maximally 2 × 10 ⁸ cells were used for the extraction. The cells were harvested and washed in 25ml 1×PBS (cold). The pellet was then resuspended in 600µl Nuclei Lysis Solution and transfered to a 2ml tube (At this step, cells could be kept in -80°C for a long time.).

The suspension was then incubated at 65°C for 15min and then cooled down to RT. 3µl RNase-Solution (RNase A, 4mg/ml) was added. The tube was kept at 37°C for 15 min. 250µlof Protein Precipitation solution was added and the suspension was vortexed. The tube was spun at full speed for 3 min in a microcentrifuge afterwards (If supernatant was not clear, it would be transferred to a new tube and spun again.).

Genomic DNA got precipitated by 700µl 100 isopropanol. The tube was centrifuged at full speed for 1 min. The pellet was washed with 600µl 70% ethanol. The DNA was dried in the air for 10-15 min and resuspended in 100-200µl of Rehydration solution at 65°C for 15 min.

A single colony picked from LB agar plate was inoculated into a test tube containing 5ml TB medium. The suspension was incubated in 37°C overnight with agitation (~250rpm). The cells were harvested the next day. Nucleospin Plasmid Kit (Macherey Nagel) was used to isolate plasmid DNA. Concentration and purity of DNA were determined by UV spectometer.

TAE buffer was used routinely for agarose gel electrophoresis in this thesis. Ethidium Bromide (0.5ug/ml in agarose gel) was used to dye DNA. UV-lamp was used to observe DNA band. DNA samples were mixed with 6×loading buffer before loaded in the gel. Electrophoresis was carried out at constant voltage (5V/cm).

TAE buffer (pH8.0)

Tris-acetic acid

40mM

EDTA

1mM

6 × loading buffer

Glycerol

50%(v/v)

EDTA

7.5mM

Xylenxyanol

0.4%(w/v)

Bromophenol Blue

0.4%(w/v)

Necleospin Extract Kit (Macherey Nagel) was used to purify DNA fragment from agarose gel after electrophoresis.

A standard Polymerase Chain Reaction was carried out in 50µl volume with the ingredient below (Saiki et al., 1988).

10 × PCR buffer

5µl

10 × dNTP Mixture (2mM)

5µl

50pM Forward primer

1µl

50pM Reverse primer

1µl

Template-DNA

1µl(10ng)

Taq Polymerase

1U

DDW

up to 50µl

Because of the high GC content of Chlamy gene, 1M Betain was often added in the PCR reaction.

The standard temperature cycle is decribed as below:

Step 1. 95°C

5 min

Step 2. 94°C

30s

Step 3. 56°C-70°C

30s

Step 4. 72°C

1min/kb

25 cycles between Step 2 and Step 4

Step 5. 72°C

5min

Step 6. 4°C

PCR reaction could not only be used to amplify target DNA for cloning but to detect the E. coli transformants. Cell clumps picked from colonies on the plate could be directly added in the PCR reaction as templates. PCR could be also used to introduce mutation in target genes.

Restriction enzymes were mainly purchased from NEB or MBI. Single restriction enzyme digestion or double restriction enzyme digestion were carried out after the direction of the manual.

The ratio between vector DNA and target DNA were normally 1:3, which also varied in different cases. The T4 ligase was purchased from NEB and the ligation reaction was carried out according to the instruction.

RNA synthesis kit (mMESSAGE mMACHINE^®)was purchased from Ambion and the process was carried out after manual instruction.

The BC Assay (bioinchoninic acid) was a colorimeric assay, which involved the reduction of Cu²⁺ to Cu⁺ by peptidic bounds of proteins. The bioinchoninic acid chelated Cu⁺ with high specificity to form a water soluble purple color complex. Concentration known BSA (Bovine Serum Albumin) solution was used as standard. The kit was purchased from Interchim. The assay procedures were carried out after the manual instructions.

SDS-PAGE was used to separate protein mixtures so that their relative abundances, purities and molecular weights could be determined. Mixed with detergent SDS (Sodium Dodecyl Sulphate) and 2-β-mercatoethanol, protein samples were heated to 95°C for 5 min for denaturation. Samples were driven by constant current of ~25mA for 1 hour.

Running Buffer

Tris

3.01g

Glycine

14.41g

SDS

1g

Dissolve in 1 l water

2×Sample buffer

Tris

65mM

SDS

3%(w/v)

Glycerol

30%(v/v)

β-mercatoethanol

5%(w/v)

Bromphenolblue

0.1(w/v)

To detect specific protein band, western blot was applied in this thesis. After SDS-PAGE, protein in the gel was transfered to a nitrocellulose membrane (Hybond-Amersham) by a semi-dry blotting system (Bio-Rad). The blotting process was carried out according to the manual instruction. After the transfer, the membrane was first blocked with blocking buffer and then incubated with blocking buffer containing the first antibody (4°C overnight or RT 1hour). The membrane was then washed for three times with blocking buffer, each time 10 min with constant agitation). Then blocking buffer with the second antibody was applied to the membrane (4°C overnight or RT 1hour). The membrane was washed for three times with PBS-Tween solution for two times and once with PBS solution, each time 10 min with constant agitation. After that, the membrane was transferred to detection buffer. BCIP and NTB were added. The membrane was then washed in water to stop the reaction when the bands were clear.

Blotting Buffer

Tris

48mM

Glycine

39mM

SDS

0.1%

PBS(Phosphate Buffered Saline)

NaCl

150mM

Na2HPO4

16mM

NaH2PO4

1.8mM

PBS-Tween

PBS buffer with 0.05% Tween (v/v)

Blocking Buffer

7% Milkpowder in PBS-Tween solution

Detection Buffer

Tris · HCl

100mM

NaCl

100mM

MgCl2

50mM

After Commassie Brilliant Blue staining or Silver staining, the band of target protein was cut out and sliced into 1mm× 1mm pieces. The pieces was collect in 1.5ml cup and sequentially washed with 50mM NH₄HCO₃, 50mM NH₄HCO₃ and 25%ACN, 25% ACN, 50% ACN. Every wash lasted for half an hour and the supernatant was discarded. The pieces were lyophilized for 1 hour.

The volume of the pieces was estimated and same volume of trypsinase solution (4µg in 100µl digestion buffer) was added. Then same amount of digestion buffer (50mM-100mM NH₄HCO₃) was added. The tube was kept in 37°C overnight.

The peptides of the digestion were extracted two times with 100 mM NH₄HCO₃ and one time with 40%ACN. Each extraction lasted for 1 hour and the extraction solutions volume were same as the volume of the pieces.

All the extraction fraction was collected and lyophilized afterwards.

Cells were harvested at 2,500 rpm for 5min and the pellet was resuspended in Buffer B (100ml cell culture in 1ml Buffer B). The suspension was centrifugated at 10,000g for 30min and the precipitation was discarded. The lysate supernatant was then loaded in the equilibrated column. The column was then washed two times with 3CV (column volume) Buffer C. Target protein was then washed off by adding 3 times 0.5 CV Buffer E.

Buffer B

Urea

8M

NaH2PO4

0.1M

Tris∙Cl

0.01M, pH8.0

Buffer C

Urea

8M

NaH2PO4

0.1M

Tris∙Cl

0.01M, pH6.3

Buffer E

Urea

8M

NaH2PO4

0.1M

Tris∙Cl

0.01M, pH4.5

Cells were harvested at 2,500 rpm for 5min and the pellet was resuspended in Buffer W (100ml cell culture in 1ml Buffer W). Cells were then broken by sonication. The suspension was centrifugated at 40,000g for 45 min. The supernatant was then collected and loaded in the equilibrated column. The column was then washed for 5 times with 1 CV (column volume) Buffer W. Target protein was then washed off by adding 6 times 0.5 CV Buffer E.

For regeneration of the column, the column was washed three times with 5CV Buffer R. When the column turned red, the column was overlaid with 2 ml Buffer R and kept in 4ºC for storage.

Buffer W (washing buffer):

Tris∙Cl

100 mM

NaCl

150 mM

EDTA

1 mM pH 8

Buffer E (elution buffer):

Tris∙Cl

100 mM

NaCl

150 mM

EDTA

1 mM

desthiobiotin

2.5 mM, pH 8

Buffer R (regeneration buffer):

Tris∙Cl

100 mM

NaCl

150 mM

EDTA

1 mM

HABA (hydroxy-azophenyl-benzoic acid)

1 mM, pH 8.0

Strain

Marker/Phenotype

Source

E.coli DH5α

supE44,(Φ80lacZΔM15),

hsdR17(r_K ^-,m_K ⁺). recA1,

endA1,gyrA96,thi-1,relA1,

deoR Δ(lacZYA-argF)U169

Promega

C. reinhardtii cw15 arg- A

cw15, arg7, mt(-)

Rochaix &van Dillevijn, 1982

C. reinhardtii CC1930

arg2 mt(-)

Duke University, USA

C. reinhardtii CC645

pab2 mt(-)

Duke University, USA

C. reinhardtii CC124

wild type, mt(-)

Duke University, USA

C. reinhardtii CC125

wild type, mt(+)

Duke University, USA

C. reinhardtii CC621

wild type, mt(-)

Duke University, USA

C. reinhardtii CC620

wild type, mt(+)

Duke University, USA

C. reinhardtii CC477

bald 1

Duke University, USA

C. reinhardtii CC478

bald 2 mt(+)

Duke University, USA

C. reinhardtii CC479

bald 2 mt(-)

Duke University, USA

C. reinhardtii CC48

arg2, mt(+)

Duke University, USA

C. reinhardtii CC32

pab1, mt(+)

Duke University, USA

C. fusiformis

Nils Koeger

X. laevis

G. Nagel

Plasmid

Marker

Source

pBluescript II KS(-)

lacZ', amp^R

Stratagene

pSP109

zeo^R, amp^R

Stevens et al 1995

pSP124S

zeo^R, amp^R

Purton S

pArg7.8

zeo^R, arg 7

Debuchy et al 1989

pSI103

amp^R, aph VIII

Sizova I

pGen-D-Ble

zeo^R, amp^R

Fischer et al 2001

pGEM-RE

amp^R

Schiereis T.(Regensburg)

pPhot1

amp^R

Schiereis T.(Regensburg)

pBlue Cf FCPcass

amp^R

Nils Koeger

pUCBM20

amp^R

Peter Berthold

pXX212

amp^R

Markus Heitzer

Oligonucleotides were purchased from Metabion.