Characterization of the Primary Structure and Cellular Localization of Silaffin-like Proteins in the Diatom Thalassiosira pseudonana

Scott Nolan HolmesPI: Dr. Kröger

Advisors: Dr. Poulsen, Dr. ScheffelPURA Research Report

Georgia Institute of Technology

Introduction:Brief Outline of Proposed Research:

Diatoms are photosynthetic algae that form cell walls (called frustules) made of silica and exhibit species-specific patterns on the nano-scale. Only recently insight into the molecular principles of this complex biological process has been gained due to the recent sequencing of two diatom genomes (Thalassiosira pseudonana and Phaeodactylum tricornutum) and the establishment of methods for molecular genetic engineering of diatoms (including T. pseudonana) which allows for identification and manipulation of proteins involved in silica formation. One class of proteins originally isolated from the diatom Cylindrotheca fusiformis, termed silaffins, have been shown to precipitate SiO2 nanospheres within minutes of being introduced into solutions of silicic acid at ambient temperatures. Silaffins have domains rich in serine and lysine residues, as well as bearing numerous post-translation modifications. Compared to the current modes of producing silica nanomaterials that require high temperatures and pressures, the silaffins are an attractive alternative that can be used industrially under ambient conditions.

Not all proteins involved in silica biosynthesis are known, so identifying proteins that are similar to silaffins and contain additional domains with different functionalities would be an important step in understanding how diatoms form silica frustules and possibly allowing for the manipulation of the silica structure on the nanoscale. Through an in silico search in the T. pseudonana genome database (http://genome.jgi-psf.org/Thaps3/Thaps3.home.html) for proteins containing at least one domain with silaffin-like serine and lysine composition, 89 silaffin-like proteins (SFLPs) have been identified (Scheffel, Poulsen and Kröger, unpublished data).

I originally proposed to study the intracellular localization of three of the 89 SFLPs; specifically SFLP-25, SFLP-26, and SFLP-63. Another protein, SFLP-28, was also added to list during the research. The SFLPs were chosen due to the presence of one or more predicted transmembrane domains, which may indicate that these proteins span the membrane of the silica deposition vesicle (i.e. the intracellular organelle directing silica formation). Such proteins have not yet been identified, but are supposed to have an important role in silica formation.

Intracellular localization of the SFLPS can be observed by creating fusion genes between the SFLPs and the gene encoding the Green Fluorescent Protein (GFP) then expressing the fusion genes in T. pseudonana. Fluorescent microscopy is then used to detect GFP fluorescence of the fusions SFLP-GFP and determine which of the SFLPs are associated with the silica cell wall or the silica deposition vesicle.

Proposal Research GoalsThe research focused on expressing GFP fusion proteins with SFLP-25, -26 and -63

in T. pseudonana and screening transformants for fluorescence. The progress of each protein differed slightly due to prior research:

• SFLP 25: complete gene structure analysis using RACE and RT-PCR is required.• SFLP-63: the gene structure has already been characterized using RACE PCR; the

gene will now be amplified from genomic DNA and cloned into the GFP containing diatom expression vector.

• SFLP-26: the genomic copy of this gene has been amplified already and inserted

into the GFP containing diatom expression vector and is now ready to be introduced via microparticle bombardment into T. pseudonana.

Additionally, SFLP-28 was added during the course of the research and also had previous research attributed to it:

• SFLP-28: the diatom transformation vector is constructed and ready for transformation.

Materials and MethodsCulture ConditionsT. pseudonana clone CCMP1335 was grown in artificial seawater NEPCC medium according to Kröger et al. (adapted from North East Pacific CultureCollection protocol at http://www3.botany.ubc.ca/cccm/NEPCC/esaw.html) with 100µg/mL of streptomycin antibiotic at 18°C and constant light of 30-35 µmol/photons·m-2·s-1. Note that the nitrogen source is 0.55 mM NaNO3 in “nitrate” medium and changed to 0.55 mM NH4CI in “ammonium” media.

Construction of Fusion SFLP-GFP Diatom Transformation VectorsPoulsen, Kröger et al. already established inducible expression vectors for

diatoms of the form pTpNR+fcp/nat+GFP. The T. pseudonana Nitrate Reductase (tpNR) promoter/terminator cassette allows expression in the presence of nitrate and no expression in the presence of ammonium. Restriction sites exist around the cassette and the eGFP gene so that other sequences may be cloned into the vector. The vector also contains the fcp+nat antibiotic resistance gene for selection. The following details the construction of each diatom transformation vector (of the form pTpNR-SFLP-GFP+fcp/nat).

pTpNR- SFLP-63-GFP+fcp/natpTpNR-GFP+fcp/nat vector was digested with EcoRV and KpnI, heat inactivated

at 80°C, treated with Calf Intestinal Alkaline Phosphatase, heat inactivated again at 75°C, and finally run on a 1.5% agarose gel in order to elute the approx. 7.5 kb vector vector. To prepare the SFLP-63 for insertion into the pTpNR vector, 3’ and 5’ RACE PCR was performed to determine the transcript boundaries. Then SFLP-63 was PCR amplified from T. pseudonana gDNA by Phusion DNA polymerase using primers SFLP63-11 5’-ATC ATA ATC ATG ATG CAG ACA AGG GCA GC -3’ and SFLP63-12 5’-AAC GAG TTG CCT TCT TGT GGG T -3’. The SFLP-63 PCR product was phosphorylated with T4 PNK. T4 ligase was used to ligate the pTpNR vector fragment and SFLP-63 PCR product together. The fusion pTpNR-SFLP-63-GFP+fcp/nat vector was then transformed into DH5α E. coli and screened for orientation on Luria-Broth plates supplemented with Ampicillin.

pTpNR- SFLP-26-GFP+fcp/natpTpNR-GFP+fcp/nat vector was digested with EcoRV, heat inactivated at 80°C,

treated with Calf Intestinal Alkaline Phosphatase, heat inactivated again at 75°C, and finally run on a 1.5% agarose gel in order to elute the approx. 7.5 kb vector vector. To prepare the SFLP-26 for insertion into the pTpNR vector, 3’ and 5’ RACE PCR was performed to determine the transcript boundaries. SFLP-26 was PCR amplified using from T. pseudonana gDNA by Phusion DNA polymerase using primers SFLP26FL-5 5’- GAT CGA TAT CAT AAT CAT GAA GTT TCC GCT CGC C-3’ and SFLP26-7 5’- GTT

CGA TAT CTT GGA CTT CTC CCT CTT TAG C-3’. The primers used to amplify SFLP-26 introduced EcoRV sites and thus the PCR product was digested with EcoRV and the corresponding 2.2 kb product was eluted from a 1.5% agarose gel. T4 ligase was used to ligate the pTpNR vector fragment and 2,2 kb SFLP-26 fragment together. The fusion pTpNR-SFLP-26-GFP+fcp/nat vector was then transformed into DH5α E. coli and screened for orientation on Luria-Broth plates supplemented with Ampicillin.

pTpNR- SFLP-28-GFP+fcp/natPrevious work by Poulsen existed on SFLP-28, namely performing 3’ and 5’

RACE PCR to map the gene boundaries and construction of the fusion gene. The pTpNR-GFP+fcp/nat vector was digested with EcoRV and KpnI, heat inactivated at 80°C, treated with Calf Intestinal Alkaline Phosphatase, heat inactivated again at 75°C, and finally run on a 1.5% agarose gel in order to elute the approx. 7.5 kb vector. SFLP-28 was PCR amplified by Phusion DNA polymerase using primers SFLP28-8 5’-ATC ATA ATC ATG AAC CTC CAA CGA GCC ATT G-3’ and SFLP28-9 5’-GTT GAG GAT ACA GTT GAT TTG CAA ATC AAC TGT ATC CTC AAC-3’. The SFLP-63 PCR product was phosphorylated with T4 PNK. T4 ligase was used to ligate the pTpNR vector fragment and 5.6 kb SFLP-28 fragment together. The fusion pTpNR-SFLP-28-GFP+fcp/nat vector was then transformed into DH5α E. coli and screened for orientation on Luria-Broth plates supplemented with Ampicillin.

Microparticle Bombardment and Selection of Transformants (adapted from Poulsen, Kröger et al.)DNA was delivered to T. pseudonana cells by using a Biolistic PDS-1000/He delivery system (BIORAD, Hercules, CA, USA). Ten mg of Tungsten particles (M5, M10, or M17) were washed with ethanol and water then resuspended in 150 µl sterile water. Aliquots of 50 µl of particles were coated under constant vortexing by adding in sequence: 5 µg of plasmid DNA, 50 µl 2.5M CaCI2, and 20 µl of 0.1 M Spermidine. T. pseudonana was grown to a cell density of 106 cells·mL-1 and centrifuged at 3000g for 10 minutes. A quantity of 108 cells were plated on NEPCC-Agar plates (1.5% agar) in a 5 cm diameter circle. Cells were dried in a sterile clean bench. Rupture disks were prepared by coating each disk with 15 µl of the particle suspension (600 ng DNA-coated particles) and drying in the clean bench. The bombardment process then started by placing a cell plate 7 cm below the stopping screen in the Biolistic chamber and affixing rupture disks into the Biolistic system. Bombardment took place at 1350 and 1550 psi for the appropriate rupture disks (disks are pressure-sensitive). After particle bombardment the cells were scrapped off by adding 2 mL of NEPCC medium and using a 1000 µl pipette tip at a low angle to carefully scrape the cells. All cells from the different pressure shootings of a certain transformant were then transferred into one liquid NEPCC culture at a ratio of 100 mL per each plate (i.e. 2 shootings for one transformant would be added to a fresh 200 mL flask with NEPCC medium) with no antibiotic. Incubate the cells for 24 hours and then plate five million cells per NEPCC agar plate (plates are supplemented at 100 µg/mL ClonNAT). Colonies were checked for fluorescence after 6-8 days of incubation.

SDS Extraction of SFLP-26Fluorescent transformants of pTpNR- SFLP-26-GFP+fcp/nat were grown in NEPCC

medium for 8-10 days. 50 mL of culture were harvested by centrifuging at 10 min max rpm (approx. 4000 rpm), pouring off the supernatant, and transferring to a 2 mL Eppendorf tube. The cells were then centrifuged briefly to remove excess liquid and resuspended in 1 mL SDS solution (2% SDS, 0.1 M EDTA, 1mM PMSF). The cells were incubated at 55°C for 30 minutes, washed three times with 500 µl of wash solution (20 mM EDTA, 1 mM PMSF), and used directly for microscopy.

SDS Extraction of SFLP-26 using CaCI2

Fluorescent transformants of pTpNR- SFLP-26-GFP+fcp/nat were grown in NEPCC medium for 8-10 days. 50 mL of culture were harvested by centrifuging at 10 min max rpm (approx. 4000 rpm), pouring off the supernatant, and transferring to a 2 mL Eppendorf tube. 500 µl of glass beads were added and the cells vortexed for 90 seconds. The glass beads were allow to settle while on ice for 5 minutes. After the supernatant was transferred to a fresh 2 mL Eppendorf, the lysate was washed three times with buffer A (1mM CaCI2, 1mM PMSF). The cells were resuspended in 100 µl of buffer A and used for microscopy.

E. coli Transformation using strain DH5α50 µl of competent DH5α E. coli cells were mixed with 10 µl ligation reaction and set on ice for 15 minutes. To induce heat shock cells were incubated at 37°C for 90 seconds and rapidly placed on ice for 2 minutes. Under sterile conditions 500 µl of SOC medium were added to the reaction and incubated at 37°C for 45 minutes. The cells were plated on LB plates supplemented with Ampicillin.

Results

Localization of SFLP-63

Figure 1. Fluorescence Microscopy of GFP fusion protein SFLP-63 in T. pseudonana. Microscopy was done using inverted-lens UV and a 63x oil-oil immersion lens; brightfield images A and C use normal light microscopy and images B and D use a GFP filter (excitation: 488 nm, emission: 505/550 nm bandpass filter) under UV light. The localization pattern is consistent with the girdle band (unpublished observations, Poulsen, Kröger et al.). Due to the cylindrical shape of T. pseudonana, there are two views of the GFP protein residing in the girdle band: D (top arrow), a ring of green as viewed looking down on the top and bottom of the cell (known as “valve” view); B, two short parallel lines at the top and bottom of the cell corresponding to viewing the cells on the side (known as “theca” view).

Localization of SFLP-28

Figure 2. Fluorescence Microscopy of GFP fusion protein SFLP-28 in T. pseudonana. Microscopy was done using inverted-lens UV and a 63x oil-oil immersion lens; brightfield images A and C use normal light microscopy and images B and D use a GFP filter (excitation: 488 nm, emission: 505/550 nm bandpass filter) under UV light. The localization pattern is consistent with the girdle band (unpublished observations, Poulsen, Kröger et al.). B, a dividing cell is noted with the girdle bands dividing. D, a theca view of the cell (left arrow) and a valve view of the cell (right arrow). Notice in C and D that the theca view cell (left arrow) is dividing.

SFLP-25PCR of T. pseudonana gDNA versus cDNA revealed no expression of the predicted SFLP-25 gene. Figure 3 shows the bands resulting from PCR of SFLP-25 using primers that bind within a predicted exon. A PCR product is predicted when amplifying gDNA. If cDNA is amplified, then a product should arise only if the SFLP-25 gene is transcribed in mRNA. The band in figure 3 shows that the primers amplify a product in gDNA but no product is amplified in cDNA; hence, SFLP-25 is not transcribed into mRNA and cannot be tagged by the techniques mentioned in this report. For reference, the expression PCR test for a gene that is expressed in cDNA (SFLP-63) is shown alongside SFLP-25 in figure 3.

Figure 3. PCR of T. pseudonana cDNA and gDNA using primers binding within the predicted coding region of SFLP-25 (A) and SFLP-63 (B). Each gel is 1.5% agarose and GeneRuler DNA ladder (Fermentas) was loaded as a standard in lanes (A) 1 and (B) 4 for both gels. A, lane 2 is the PCR of T. pseudonana gDNA with primers (SFLP25-7 and SFLP25-8) binding to a predicted exon, while 3 and 4 represent the same reaction done on two different cDNA batches. The gDNA reaction shows a bright band around 600 bp while the cDNA reactions lack any bands (except for primer bands at the bottom of the gel). Thus, SFLP-25 is not transcribed into mRNA in T. pseudonana. B, lane 1 is the PCR of T. pseudonana gDNA with primers (SFPL63-8, SFPL63-9) binding to a predicted exon, while lanes 2 and 3 are the same PCR reaction but using two different T. pseudonana cDNA batches. Similarly, lanes 5, 6, and 7 represent PCR reactions of gDNA and two cDNA batches using primers SFLP63-9 and SFLP63-10. The appearance of discrete bands at sizes predicted by the genome model indicate expression of the SFLP-63 transcript.

SFLP-26Fluorescent microscopy of cells with GFP-tagged SFLP-26 indicated localization of the cell membrane (Scheffel, Holmes unpublished observation). However, to differentiate between the cell membrane and the frustules (cell wall), SDS extraction of the cell lysate was performed with and without CaCI2. Figure 4 shows the results of the extractions; any protein embedded or associated with the frustules will remain in the extract, which are the proteins of current interest. No GFP fluorescence occurs in the cell extract of either SDS extraction. Thus, SFLP-26 is associated with the cell membrane and not the diatom frustules.

Figure 4. SDS extraction of fluorescent T. pseudonana cells expressing SFLP-26. Diatoms cells visualized with light microscopy (left) and inverted-lens UV 63x oil immersion lens under a FITC filter (right). The lack of GFP fluorescence in the extract suggests the SFLP-26 tagged GFP protein, which was seen to localize in the cell membrane, is not associated with the diatom frustules (cell wall).

Project ConclusionThe original proposal for the SFLP project focused on the creating fusion genes and localizing three SFLPs (SFLP-25, SFLP-26, and SFLP-63); SFLP-26 localized to the cell membrane, SFLP-63 was localized to the girdle bands of T. pseudonana, and SFLP-25 was found to not be transcribed into mRNA. Another gene, SFLP-28, was also transformed into T. pseudonana and found to localize in the girdle bands. The project started before the scholarship period (Summer 2009 to Fall 2009), specifically the RACE PCR of SFLP-25 and the construction of the SFLP-26 fusion gene. The results of the project may be published in the future within Dr. Scheffel’s research on additional SFLPs.

ReferencesAppendixPrimersSFLP25-7 5’-CCA GAG AAG GAA CGA TCA GG-3’SFLP25-8 5’-CAT CTG GGT AGA AAG GTG GCC-3’

SFLP26FL-5 5’-GAT CGA TAT CAT AAT CAT GAA GTT TCC GCT CGC C-3’ SFLP26-7 5’-GTT CGA TAT CTT GGA CTT CTC CCT CTT TAG C-3’

SFLP28-8 5’-ATC ATA ATC ATG AAC CTC CAA CGA GCC ATT G-3’ SFLP28-9 5’-GTT GAG GAT ACA GTT GAT TTG CAA ATC AAC TGT ATC CTC AAC-3’

SFLP63-8

5’-GCA ACG ACC AAA CGA CGC CAC-3’SFLP63-95’-TCA GTG TGT GAC GAG TCA ATG-3’SFLP63-105’-GAA AGT TAG ACG ACT GTT GGT GA-3’SFLP63-11 5’-ATC ATA ATC ATG ATG CAG ACA AGG GCA GC -3’ SFLP63-12 5’-AAC GAG TTG CCT TCT TGT GGG T -3’