Genetic transformation

7 posts / 0 new
Last post
Rich_biotech
Rich_biotech's picture
Genetic transformation

is there a protocol for genetic transformation and gene expression of micro-algae?

Tony Rook
Tony Rook's picture
Here is a US Patent that may

Here is a US Patent that may be of some help to you...

Method to transform algae, materials therefor, and products produced thereby. United States Patent 5661017
US Patent Issued on August 26, 1997

Background of the invention:

...Over the past decade, genetic transformation has become routine for many organisms, including bacteria, yeast, mammalian cells and some higher plants. However, there has been little success mn developing transformation systems for eucaryotic microalgae, due partly to the recalcitrance of commonly-used algal species to standard transformation techniques and genetic markers. This phenomenon is likely to be due to the difficulty of introducing foreign DNA into the algal cell through the cell wall and to poor expression of commonly used transformation markers, such as neomycin phosphotransferase or other antibiotic resistance genes, by the algae. To date, the only eucaryotic microalgae for which there are reproducible transformation systems are the single-celled green alga Chlamydomonas reinhardtii and a closely related colonial species Volvox carterii. However, successful transformation of these organisms to date has required the use of homologous genes as selectable transformation markers. These protocols often require the development of auxotrophic mutants which can be transformed with plasmids containing wild-type homologous genes, rendering the cells prototrophic.

In view of the above discussion, a need exists for a genetic transformation system which is widely useful in algae.

Summary of the invention:

The present invention includes a method to transform chlorophyll C-containing algae which includes introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory DNA sequence into a chlorophyll C-containing alga in such a manner that the marker is produced by the alga. In a preferred embodiment the chlorophyll C-containing alga is a diatom and in more preferred embodiments is of a genus selected from the group consisting of Cyclotella and Navicula. In a further embodiment, the method can include introducing a recombinant molecule comprising a nucleic acid molecule encoding a product which is operatively linked to an algal regulatory control sequence into the alga such that the product is produced by the alga. In further preferred embodiments, the regulatory control sequences can include a Cyclotella cryptica acetyl-CoA carboxylase regulatory control sequence.

A further embodiment of the present invention includes a chimeric molecule which includes one or more regulatory control sequences derived from one or more chlorophyll C-containing algae operatively linked to a nucleic acid molecule encoding a selectable marker, an RNA molecule, or a protein, and wherein the nucleic acid molecule is not naturally associated with one or more of the regulatory control sequences. In a further preferred embodiment, the regulatory control sequences in the chimeric molecule are derived from a diatom, and preferably Cyclotella cryptica.

A further embodiment of the present invention includes a method to produce a recombinant chlorophyll C-containing algal strain which is transformed with a nucleic acid molecule encoding a dominant selectable marker in such a manner that the marker is produced by the strain. The method includes culturing the transformed strain in the presence of a compound that is toxic to algae not transformed by the marker nucleic acid molecule and to which the dominant selectable marker provides resistance, and subsequently isolating from the culture an algal strain that is capable of growing in the presence of the compound.

Further embodiments of the present invention include nucleic acid molecules which include nucleic acid sequences identified as SEQ ID NOS:1,2 or 3, or portions thereof having a regulatory function corresponding to the nucleic acid sequences of the SEQ ID NOS:1,2 or 3.

Tony Rook
Tony Rook's picture
Here are some articles that

Here are some articles that may help....

Verena Jung, Thierry Thibaut, Alexandre Meinesz and Georg Pohnert. Comparison of the Wound-Activated Transformation of Caulerpenyne by Invasive and Noninvasive Caulerpa Species of the Mediterranean. Journal of Chemical Ecology. Volume 28, Number 10 / October, 2002. 2091-2105
DOI: 10.1023/A:1020710230532

Abstract:
The invasive green alga, Caulerpa taxifolia, that has spread rapidly after its introduction into the Mediterranean and the North American Pacific, reacts to wounding by transforming its major metabolite caulerpenyne (1). This wound-activated reaction involves the transformation of the bis-enol acetate moiety of 1, releasing reactive 1,4-dialdehydes. The ability to perform this transformation is found also in both the noninvasive Mediterranean C. prolifera and the invasive C. racemosa. Trapping experiments, as well as transformation of the model substrate geranyl acetate, suggest that all three investigated Caulerpa spp. rely on esterases that act upon wounding of the algae by subsequently removing the three acetate residues of caulerpenyne. The resulting reactive 1,4-dialdehyde oxytoxin 2 (9) can be identified by liquid chromatographymass spectrometry and is unstable in the wounded tissue. Caulerpenyne transformation occurs rapidly, and severe tissue damage caused degradation of more than 50% of the stored caulerpenyne within 1 min in all three algae. Prevention of the enzymatic reaction before extraction, by shock freezing the tissue with liquid nitrogen, was used for the determination of the caulerpenyne content in intact algae. It gives about twofold higher values compared to an established methanol extraction protocol. The speed and mechanism of the wound-activated transformation, as well as the caulerpenyne content in intact tissue of invasive and noninvasive Caulerpa spp., are comparable. Thus, this enzymatic , transformation, despite being fast and efficient, is likely not the key for the success of the investigated invasive species.

_________________________________________________________

A. Hallmann and S. Wodniok. Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa. Plant Cell Reports. Volume 25, Number 6 / June, 2006. 582-591
DOI: 10.1007/s00299-006-0121-x

Abstract:
Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5′ and 3′ flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with 3′ and 5′ untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist.

Rich_biotech
Rich_biotech's picture
thnx all. that was great help

thnx all. that was great help

Tony Rook
Tony Rook's picture
And here are some more

And here are some more references which should have some good protocols for transformation of algae...

Sasidharanpillai Vinod Kumar, Rachel William Misquitta, Vanga Siva Reddy, Basuthkar Jagadeeswar Rao and Manchikatla Venkat Rajam. Genetic transformation of the green algaChlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Science. Volume 166, Issue 3, March 2004, Pages 731-738
doi:10.1016/j.plantsci.2003.11.012

Abstract:
We report here the first successful transfer of T-DNA of Agrobacterium tumefaciens carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of the green alga Chlamydomonas reinhardtii. The transformation frequency (which was based on hygromycin resistance phenotype) was 50-fold higher than that of the glass bead transformation. Molecular and genetic analyses performed on transformants revealed the stable nuclear integration and expression of transgenes. The simplicity of Agrobacterium-mediated gene transfer and the high transformation frequency as well as the precision of T-DNA integration will enable further molecular dissection of this important model organism to understand basic plant metabolic processes as well as to exploit the system for biotechnological applications.

________________________________________________________

Miri Lapidot, Dina Raveh, Alex Sivan, Shoshana (Malis) Arad, and Michal Shapira. Stable Chloroplast Transformation of the Unicellular Red Alga Porphyridium Species. Plant Physiol, May 2002, Vol. 129, pp. 7-12

Abstract:
Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalga Porphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.

______________________________________________________

Stephen P. Mayfield, Scott E. Franklin, and Richard A. Lerner. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci U S A. 2003 January 21; 100(2): 438442.
doi: 10.1073/pnas.0237108100.

Abstract:
Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5′ and 3′ RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5′ and 3′ elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

_______________________________________________________

Vladimir G. Ladygin. Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation. Process Biochemistry. Volume 39, Issue 11, 30 July 2004, Pages 1685-1691
doi:10.1016/j.procbio.2003.07.001

Abstract:
Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 106 cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 103 hygromycin-resistant clones of trasformants per 106 recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated.

____________________________________________________________

Good luck! When you get a chance let us know which protocol worked best for you application.

drsalihin
drsalihin's picture
Sir, I have an intense

Sir, I have an intense longing to produce biodiesel from algea using genetic transformation. shall you help me?

SIUEGrad
SIUEGrad's picture
Tony Rook. Did we ever hear

Tony Rook. Did we ever hear from Rich_biotech about what protocol worked best for him?