DNA constructs are generated in order to introduce designer butanol-production­pathway genes to a host alga, plant, plant tissue, or plant cells. That is, a nucleotide sequence encoding a designer butanol-production-pathway enzyme is placed in a vector, in an operable linkage to a promoter, preferably an inducible promoter, and in an operable linkage to a nucleotide sequence coding for an appropriate chloro- plast-targeting transit-peptide sequence. In a preferred embodiment, nucleic acid constructs are made to have the elements placed in the following 5′ (upstream) to 3′ (downstream) orientation: an externally inducible promoter, a transit targeting sequence, and a nucleic acid encoding a designer butanol-production-pathway enzyme, and preferably an appropriate transcription termination sequence. One or more designer genes (DNA constructs) can be placed into one genetic vector. An example of such a construct is depicted in Fig. 2a. As shown in the embodiment illustrated in Fig. 2a, a designer butanol-production-pathway transgene is a nucleic acid construct comprising: (a) a PCR forward primer; (b) an externally inducible promoter; (c) a transit targeting sequence; (d) a designer butanol-production-path­way-enzyme-encoding sequence with an appropriate transcription termination sequence; and (e) a PCR reverse primer.

In accordance with various embodiments, any of the components (a)-(e) of this DNA construct are adjusted to suit for certain specific conditions. In practice, any of the components (a)-(e) of this DNA construct are applied in full or in part, and/or in any adjusted combination to achieve more desirable results. For example, when an algal hydrogenase promoter is used as an inducible promoter in the designer butanol — production-pathway DNA construct, a transgenic designer alga that contains this DNA construct will be able to perform autotrophic photosynthesis using ambient air CO2 as the carbon source and grows normally under aerobic conditions, such as in an open pond. When the algal culture is grown and ready for butanol production, the designer transgene(s) can then be expressed by induction under anaerobic condi­tions because of the use of the hydrogenase promoter. The expression of designer gene(s) produces a set of designer butanol-production-pathway enzymes to work with the Calvin cycle for photobiological butanol production (Fig. 1).

The two PCR primers are a PCR forward primer (PCR FD primer) located at the beginning (the 5′ end) of the DNA construct and a PCR reverse primer (PCR RE primer) located at the other end (the 3′ end) as shown in Fig. 2a. This pair of PCR primers is designed to provide certain convenience when needed for relatively easy PCR amplification of the designer DNA construct, which is helpful not only during and after the designer DNA construct is synthesized in preparation for gene trans­formation, but also after the designer DNA construct is delivered into the genome of a host alga for verification of the designer gene in the transformants. For example, after the transformation of the designer gene is accomplished in a C. reinhardtii — arg7 host cell using the techniques of electroporation and argininosuccinate lyase (arg7) complementation screening, the resulted transformants can be then analyzed by a PCR DNA assay of their nuclear DNA using this pair of PCR primers to verify whether the entire designer butanol-production-pathway gene (the DNA construct) is successfully incorporated into the genome of a given transformant. When the nuclear DNA PCR assay of a transformant can generate a PCR product that matches with the predicted DNA size and sequence according to the designer DNA construct, the successful incorporation of the designer gene(s) into the genome of the transfor­mant is verified.

Therefore, the various embodiments also teach the associated method to effec­tively create the designer transgenic algae, plants, or plant cells for photobiological butanol production. This method, in one of embodiments, includes the following steps: (a) selecting an appropriate host alga, plant, plant tissue, or plant cells with respect to their genetic backgrounds and special features in relation to butanol produc­tion; (b) introducing the nucleic acid constructs of the designer genes into the genome of said host alga, plant, plant tissue, or plant cells; (c) verifying the incorporation of the designer genes in the transformed alga, plant, plant tissue, or plant cells with DNA PCR assays using the said PCR primers of the designer DNA construct; (d) measuring and verifying the designer organism features such as the inducible expression of the designer butanol-pathway genes for photosynthetic butanol production from carbon dioxide and water by assays of mRNA, protein, and butanol-production characteris­tics according to the specific designer features of the DNA construct(s) (Fig. 2a).

The above embodiment of the method for creating the designer transgenic organ­ism for photobiological butanol production can also be repeatedly applied for a plurality of operational cycles to achieve more desirable results. In various embodiments, any of the steps (a)-(d) of this method described earlier are adjusted to suit for certain specific conditions. In various embodiments, any of the steps

(a) -(d) of the method are applied in full or in part, and/or in any adjusted combina­tion. Many examples of designer butanol-production-pathway genes (DNA con­structs) are shown in the sequence listings: SEQ ID NOS:1-57 of the PCT Patent Application Publication No. WO 09105733 and SEQ ID NOS:1-165 of the US Patent Application Publication No. 2011/0177571 A1.

The nucleic acid constructs, such as those presented in the examples earlier, may include additional appropriate sequences, for example, a selection marker gene, and an optional biomolecular tag sequence (such as the Lumio tag). Selectable markers that can be selected for use in the constructs include markers conferring resistances to kanamycin, hygromycin, spectinomycin, streptomycin, sulfonyl urea, gentamy — cin, chloramphenicol, among others, all of which have been cloned and are avail­able to those skilled in the art. Alternatively, the selective marker is a nutrition marker gene that can complement a deficiency in the host organism. For example, the gene encoding argininosuccinate lyase (arg7) can be used as a selection marker gene in the designer construct, which permits identification of transformants when C. reinhardtii arg7- (minus) cells are used as host cells.

Nucleic acid constructs carrying designer genes can be delivered into a host alga, blue-green alga, plant, or plant tissue or cells using the available genetic transforma­tion techniques, such as electroporation, PEG-induced uptake, and ballistic delivery of DNA, and Agrobacterium-mediated transformation. For the purpose of deliver­ing a designer construct into algal cells, the techniques of electroporation, glass bead, and biolistic gene gun can be selected for use as preferred methods, and an alga with single cells or simple thallus structure is preferred for use in transforma­tion. Transformants can be identified and tested based on routine techniques.