Genetic Engineering

 

A significant body of research has been devoted to the production of biodiesel from economic feedstock resources mainly algae. Various approaches have been applied to increase the oil productivity of algae. From these approaches, genetic engineering emerges as one of the most significant tools for the overproduction of lipids from microalgae. The enzyme Acetyl CoA Carboxylase (ACCase) was found to catalyze a key metabolic step in the synthesis of oils in algae. This enzyme has been already isolated from a diatom. The gene that encodes for the production of ACCase was eventually isolated and cloned. With this gene in hand, it will be possible to develop a successful transformation system for algae (particularly diatoms) as tools and genetic components for expressing a foreign gene.

 

R&D Center and Genetic Engineering:

The main objective is to produce recombinant microorganisms (E. coli, yeast fungi and some algal spp.) able to produce high amounts of fatty acids and triglycerides as feedstock for biodiesel industry. The genetically engineered (GE) microorganisms must be tested and approved to be capable of overproduction of biodiesel feedstock lipids. The R&D research plan includes the following two stages:

 

First Stage:

  1. Design of degenerate primers specific for the Acetyl CoA Carboxylase  gene based on the amino acid sequences for this gene (conservative regions), which cited on the Gene Bank (achieved).

  2. Isolation of Acetyl CoA Carboxylase gene (or part of the gene) using the degenerate primers and using the amplified fragment as a probe (achieved).

  3. Isolation of the Genomic DNA from the algal cells (or Rizobia, Sunflower plant, Canola or whatever plant), followed by construction of genomic library (achieved).

  4. Using the amplified gene as a probe for screening the recombinant clones with hybridization techniques (Ongoing).

  5. Sub-cloning for the recombinant gene (sole gene) into a prokaryotic expression vector, in vitro transcription for that gene (Ongoing).

  6. Studying the expression of the gene and the amount of the fatty acids and triglycerides produced (the type of the fatty acids also should be considered) (Near Future).

  7. If the amount of the Fatty acids produced is not enough, the gene will be put under constitutive promoter to increase the fatty acid production (Near Future).

 

Second Stage:  (Current reseraches)

  1. Sub-cloning the gene into Eucharistic expression vector.

  2. Transformation the cassette into algal cells (of different algal species) using electroporation technique.

  3. Evaluation of the recombinant algal cells after being cultured on suitable media, and examine the transformation efficiency using the marker genes in the cassette (the constructed gene) as indicators for successful recombination and to assure whether or not the constructed gene (of the cassette) was integrated on the algal genome.

  4. Evaluation (expressed as overproduction of algal fats) of the amount of fatty acids and triglycerides produced by the recombinant algae.