Hydrogen production by means of a cell expression system
A technology of hydrogen and cells, applied in biochemical equipment and methods, enzymes, redox enzymes, etc., can solve the problems of hydrogen production on an industrial scale
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[0166] Preparation of Hox expression vector
[0167] Those skilled in the art will be aware of the molecular techniques available for expression vector preparation.
[0168] Nucleic acid molecules for incorporation into expression vectors of the invention as described above can be prepared by synthesizing the nucleic acid molecules using oligonucleotides that serve as primers to each other and the nucleic acid sequences described herein.
[0169] A number of molecular techniques have been developed for operably linking DNA to vectors via complementary cohesive ends. In one embodiment, a band of complementary homopolymers can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then ligated by hydrogen bonding between the complementary homopolymeric tails to form a recombinant DNA molecule.
[0170] In alternative embodiments, synthetic linkers containing one or more restriction sites provided are used to operabl...
Embodiment 1
[0223] Construction of expression vector
[0224] The Synecocystis PCC 6803 library was used as template and oligonucleotide primers SynBamFwd: ccaatcatgg atccgctgta ttgctccttt ttgagg (SEQ ID NO: 14) and SynEcoRev: ggattactga attcccgtct gaatgttttt tg (SEQ ID NO: 15) were generated by PCR amplification. The coding region of the bidirectional hydrogenase protein complex is SEQ ID NO:1. The resulting gene sequence encodes SEQ ID NO: 1, which includes BamHI and EcoRI restriction sites incorporated at the 5' and 3' ends, respectively.
[0225] As shown in Figure 4, the resulting PCR product was cleaved at the incorporated restriction site by restriction enzymes, BamHI and EcoRI, and inserted by ligation using T4 ligase, which had also been restricted using BamHI and EcoRI. The cleaved expression vector pET-17b (described previously) was digested with a sex endonuclease.
Embodiment 2
[0227] Construction of expression vector
[0228]In an alternative example, using the Synechocystis PCC 6803 library as template and oligonucleotide primers SynBamFwd: ccaatcatgg atccgctgta ttgctccttt ttgagg (SEQ ID NO: 14) and SynNotRev: ggattactgc ggccgcccgt ctgaatgttt tttg (SEQ ID NO: 16) by PCR amplification generates the coding region of the bidirectional hydrogenase protein complex, namely SEQ ID NO:1. The resulting gene sequence encodes SEQ ID NO: 1, which includes BamHI and NotI restriction sites incorporated at the 5' and 3' ends, respectively.
[0229] The resulting PCR product was cleaved by restriction enzymes, namely BamHI and NotI, at the incorporated restriction sites, and using T4 ligase, was inserted by ligation into the DNA that had been cleaved by restriction endonuclease digestion with BamHI and NotI. Expression vector pET-17b (described previously).
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