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General

Yes, please see the table below comparing the two algae organisms we offer:

 

Synechococcus

Chlamydomonas

Organism

Synechococcus elongatus Pcc7942

Chlamydomonas reinhardtii 137c

Cell type

Prokaryotic—Cyanobacterium (blue-green algae)

Eukaryotic—unicellular green algae

Genome size

2.7 Mb

121 Mb

Media

BG11—for Cyanobacteria in general

TAP—for Chlamydomonas only

Integration

At Neutral Site 1

Random

Selection for plasmid propagation in E. coli

Spectinomycin (100 μg/mL)

Ampicillin (100 μg/mL)

Promoter for GOI

Psc = weak constitutive promoter* (pSyn_1 vector) or PpsbA = strong constitutive promoter (pSyn_6 vector)

Hsp70A-RbcS2 = strong, constitutive (Hsp 70 and RbcS2 hybrid)

Selection for integration

Spectinomycin (10 μg/mL)

Hygromycin (10 μg/mL)

Transform algae with

Supercoiled plasmid

Linearized plasmid (such as xScaI)

RBS in TOPO vector

Yes

No

RBS in RE vector

No (pSyn_1 vector)
Yes (pSyn_6 vector)

No

Potential for silencing

Has not been reported

Can be a problem, dependent on the gene sequence and protein toxicity; pChlamy_4 vector is designed so that proteins are expressed as transcriptional fusion with sh-ble, providing higher expression levels. The Zeocin selection helps to maintain gene expression during passage.

ATG sequence

No start codon (from the native gene or add to primer) in pSyn_1 vector; ATG initiation codon at NdeI cloning site in pSyn_6 vector

Already provided in the vector (Intro-1 Rbc S2)

Transcription termination

rrnB = strong

None

UTR

 

3’ UTR after the MCS (pChlamy_3 and pChlamy_4)**

* The original pSyn_1 vector contained the PNI promoter, which was induced by nickel; however, when too much nickel was added, it became toxic to the cells, and therefore, the promoter was changed to Psc. Nickel induction is no longer recommended.
**The original version, pChlamy_1 vector, does not have a 3’ UTR and should be added to your insert immediately following the stop codon for expressing high levels of recombinant protein.

Graph A below depicts the enzyme xylanase cloned into vector pChlamy_4, and transformed into Chlamydomonas reinhardtii 137C. Activity was measured by EnzChek® Ultra Xylanase Assay Kit, and was 17.6-fold higher than that observed with previous systems. Graph B depicts the gene for B-glucuronidase (GUS) cloned into vector pSyn_6 and transformed into Synechococcus elongatus. GUS activity levels were >100-fold higher than those seen in our previous system.

The average expected time constant is 15–20 milliseconds. We have a Neon® electroporation protocol in the manual.

Yes, unfortunately, silencing is a big problem in algae. The extent of silencing depends on the sequence of the gene and the toxicity of the protein that expresses from that gene to the cell.

The engineering kit is designed for random insertion of your gene of interest, which can lead to your gene being lost quickly. The protein expression kit, which contains the pChlamy_4 vector, is designed so that proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.

Unfortunately, we have not tested TAP media with other kinds of green algae. There is an indication that Chlorella will grow if the TAP media is vitamin-fortified, but we don’t have the specifics.

We offer premade TAP growth media optimized for Chlamydomonas (Cat. No. A1379801). Please see the formulation below:
TAP Media

Item

Formula

(% w/v)

EDTA (Disodium Salt)

EDTA

0.0005%

Zinc Sulphate Heptahydrate

ZnSO4·7H2O

0.002%

Boric Acid

H3BO3

0.001%

Manganese Chloride
Tetrahydrate

MnCl2·4H2O

0.005%

Cobalt Chloride
Hexahydrate

CoCl2·6H2O

0.002%

We offer ready-to-use BG-11 Media, optimized for Cyanobacteria (Cat. No. A1379901). There are several BG11 media formulations. Please see our formulation below.
Our BG-11 media have been qualified for culturing Synechococcus elongatus (strain PCC 7942).

Item

Formula

(%w/v)

Boric Acid

H3BO3

0.00287%

Manganese Chloride
Tetrahydrate

MnCl2·4H2O

0.00181%

Zinc Sulphate
Heptahydrate

ZnSO4·7H2O

0.00022%

Sodium Molybdate
Dihydrate

Na2MoO4

0.00039%

Copper Sulphate
Pentahydrate

CuSO4·5H2O

0.00008%

Sodium Nitrate

NaNO3

0.15000%

Calcium Chloride
Dihydrate

CaCl2.·2H2O

0.00270%

Ferric Ammonium Citrate
(Green)

C6H5+4yFexNyO7

0.00120%

EDTA

EDTA

0.00010%

Potassium Phosphate
Dibasic

K2HPO4

0.00390%

Magnesium Sulfate
Heptahydrate

MgSO4·7H2O

0.00750%

Sodium Carbonate
Monohydrate

Na2CO3·H2O

0.00200%

GeneArt® gene synthesis was used to create the plasmid backbones synthetically. Also, GeneArt® is the umbrella brand for synthetic biology, which is a large area in which these kits will be used.

The cell growth can be measured by OD750 and the linear range will be between 0.2 and 1.2 (in 1 cm lightpass). If too high, please dilute them and measure again. It usually takes 5–6 days for full growth. An accurate measure can be taken using the Countess® Cell Counter or Tali® Cytometer from Life Technologies™. The formula to calculate cell number is as follows:

Cell concentration (cells/mL) = (OD750 – 0.088)/9 × 108  = (OD750 – 0.088)/(9 × 10-8)

Please note, our COA states that “240 μl of frozen cells are thawed out and cultured in 6 mL of Gibco® TAP media at 28°C and 50 μE light. Optical density is measured at 750 nm following 6 days of growth. Optical density must reach 0.6. Viability testing is performed on a minimum of 6 vials per lot.”

We recommend our Synechococcus kits, where the integration is directed to the Neutral Site 1 of the algae genome.

Chlamydomonas Protein Expression

After 6 months of storage, the Chlamydomonas cells did not lose their competency. It is expected that they may with longer storage times, but we have yet to gather data points for loss of competency after one year of storage. 

The strain is Chlamydomonas reinhardtii 137c. This is considered to be a wild type lab strain, mating type “mt +.”

There are both cell wall minus and positive strains; 137c has a cell wall.

The Chlamydomonas cells should be dark green in color. Light green or even colorless (and maybe some droplets along the side of the vial) cells are indicative of freeze/thawing or fluctuations in temperature, which Chlamydomonas cells are extremely sensitive to.  

The kits are not designed for long-term storage of positive-selected clones. Most people keep the plates on their bench top at room temperature (not 4°C, as they need light) while in use, re-streaking if necessary. The GeneArt® Cryopreservation Kit for Algae can be used to preserve algal strains and clones for storage at –80°C for years.

Transfection with these vectors is a stable transfection.

Integration is at random. The engineering kit is based on a random insertion in which the gene of interest can be lost very quickly. For the protein expression kit, the pChlamy_4 vector is designed so proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.

We don’t report transformation efficiency of the Chlamydomonas cells, since the end result of transformation is random integration into the genome. The electroporation results will depend on the gene of interest. The control vector should produce a minimum of 30 transformants per electroporation reaction. The number of positive clones per colonies picked should be ≥90% with your gene of interest.

The expected time constant is 15–20 milliseconds (average is 17 milliseconds).

After 6 months of storage, the Chlamydomonas did not lose their competency. We have currently not tested beyond this time point.

Yes, if you plan to use the pChlamy_1 vector to express high levels of recombinant protein, your insert also needs to contain a 3’ UTR (untranslated region) immediately following the stop codon.

Yes, the pChlamy_3 vector contains a 3’ UTR after the multiple cloning site.

Yes. For the TOPO version, you can design the primer to make sure the coding sequence is in frame with the ATG in the vector.

The pChlamy_1 vector does not have the stop codon and the 3' UTR. The pChlamy_3 vector has the 3’UTR that has been shown to increase protein expression.

The pChlamy_4 vector was designed in order to circumvent the transgene silencing that often occurs in C. reinhardtii. This vector is also designed so that proteins are expressed as transcriptional fusions with the blemoycin/zeocin resistance gene (sh-ble). The self-cleaving sequence for the 2A peptide from the foot-and-mouth-disease-virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.

Synechococcus Protein Expression

Synechococcus elongatus strain PCC 7942, a model cyanobacterium, is used.

A double homologous recombination event occurs between the Neutral Site 1 in the genome and between NSa and NSb in the plasmid. This results in the gene of interest and spectinomycin resistance integrating into the genome within the Neutral Site 1.  

After 6 months of storage, the Synechococcus did not lose their competency. We have not yet tested longer storage points.

The promoter for pSyn_1 is the Psc. Psc is a weak constitutive promoter that drives basal expression of your gene of interest. The promoter was taken from the solanesyl diphosphate synthase gene from Synechocystis sp. Strain PCC 6803. Note: Use of this weak constitutive promoter is a good fit for applications that are hindered by strong expression, such as pathway engineering or complementation of mutant genes normally expressed at low levels (Simkovsky et al., 2012).

The promoter for pSyn_6 is the psbA promoter (PpsbA). PpsbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving high-level expression of your gene of interest. The pSyn-6 vector also includes a ribosome-binding site (GAAGGAG) for efficient initiation of translation, as well as a stop codon. A 6xHis TEV and a V5-His epitope tag is also included in the vector backbone for detection/purification of the gene of interest. NS1 (neutral site 1) homologous recombination sites are present in both vectors for the integration of the vector into the Synechococcus elongatus genome.

Cyropreservation

Yes, we offer our GeneArt® Cryopreservation Kit for Algae (Cat. No. A24228).

Simply grow your cells in the presence of Cryopreservatin Reagent A for 2–5 days, harvest the cells, and resuspend them in Cryopreservation Reagent B. Follow by freezing the cells.

We have tested the kit with Chlamydomonas and Chlorella strains, showing a 5% and 100% cell viability, respectively.

We have used the following freezing containers with this product:

  • Thermo Scientific™ Mr. Frosty® Freezing Container
  • Biocision™ CoolCell™ FTS30 Cell Freezing Container

Transformation Reagent

We offer our MAX Efficiency® Transformation Reagent for Algae (Cat. No. A24229). Eight different strains of Chlamydomonas reinhardtii, CC1690, CC2935, CC1009, CC4414, CC118, CC536, WT137c, and CC3395 wall(-), were transformed using MAX Efficiency ® Transformation Reagent for Algae and with TAP/sucrose reagent by electroporation. The comparison below shows that MAX Efficiency® Transformation Reagent for Algae resulted in >1000-fold colonies (right) over TAP/sucrose media in C. reinhardtii WT137c (below).