They used wild-type A.Victoria GFP, and exchanged 65 Serin to Try.
The patent mentions using promoters other than native GFP promoter
e.g. here
3. The method of claim 2 wherein means for effecting expression of said polynucleotide is E. coli strain BL21 (DE3)Lys S transformed with an expression vector comprising, 5' of said one or more sequences of nucleotide bases collectively encoding the amino acid sequence of pre-coelenterazine peptide, one or more appropriate regulatory control sequences which collectively enable expression of said polynucleotide; and one or more sequences of bases which collectively confer resistance to an antibiotic upon an organism.
4. The method of claim 3 wherein said means for effecting expression of said polynucleotide is the E. coli SMC2 (ATCC Accession No. 69553).
It seems they used the cDNA
To generate a polynucleotide having these encoding sequences, one may introduce one or more point mutations into the cDNA for the GFP of A. victoria in the cDNA described in Prasher, et al., supra, using in vitro mutagenesis methods well known to those skilled in the art.
Btw.,
Parent Case Data: This application is a division of application Ser. No. 08/192,158, filed Feb. 4, 1994 now abandoned.
Patent invalid?
Oh god,
This code is degenerate; thus, the Arg at residue 109 may be encoded by nucleotide bases as CGT, but could, under the code, equally be CGC, CGA, CGG, AGA or AGG, and still encode for Arg. Similarly, nucleotide bases encoding Tyr as TAT could equally be TAC and still encode Tyr for residue 65 in the peptide. Alternatively, the polynucleotide may be a cDNA (or RNA equivalent) which includes, in addition to the nucleotides for Ser65 being altered from TCT to TAT, mu
It feels like 90% of the patent explained something obvious for people who are a bit familiar with biology....
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